Medically Relevant Acinetobacter Species Require a Type II Secretion System and Specific Membrane-Associated Chaperones for the Export of Multiple Substrates and Full Virulence

Acinetobacter baumannii, A. nosocomialis, and A. pittii have recently emerged as opportunistic human pathogens capable of causing severe human disease; however, the molecular mechanisms employed by Acinetobacter to cause disease remain poorly understood. Many pathogenic members of the genus Acinetobacter contain genes predicted to encode proteins required for the biogenesis of a type II secretion system (T2SS), which have been shown to mediate virulence in many Gram-negative organisms. Here we demonstrate that Acinetobacter nosocomialis strain M2 produces a functional T2SS, which is required for full virulence in both the Galleria mellonella and murine pulmonary infection models. Importantly, this is the first bona fide secretion system shown to be required for virulence in Acinetobacter. Using bioinformatics, proteomics, and mutational analyses, we show that Acinetobacter employs its T2SS to export multiple substrates, including the lipases LipA and LipH as well as the protease CpaA. Furthermore, the Acinetobacter T2SS, which is found scattered amongst five distinct loci, does not contain a dedicated pseudopilin peptidase, but instead relies on the type IV prepilin peptidase, reinforcing the common ancestry of these two systems. Lastly, two of the three secreted proteins characterized in this study require specific chaperones for secretion. These chaperones contain an N-terminal transmembrane domain, are encoded adjacently to their cognate effector, and their disruption abolishes type II secretion of their cognate effector. Bioinformatic analysis identified putative chaperones located adjacent to multiple previously known type II effectors from several Gram-negative bacteria, which suggests that T2SS chaperones constitute a separate class of membrane-associated chaperones mediating type II secretion.


Introduction
Members of the genus Acinetobacter are regarded as opportunistic human pathogens of increasing relevance worldwide due in part to the rapid emergence of multiply-drug resistant strains [1].In fact, the Center for Disease Control and Prevention has recently categorized multi-drug resistant Acinetobacter at the serious hazard level, prompting sustained research and action to further prevent its dissemination.Specifically, A. baumannii, A. pittii, and A. nosocomialis of the Acinetobacter calcoaceticus-baumannii (Acb) complex have become the most medically relevant members of the genus as they are most frequently isolated from health care facilities as well as human tissues [2].Although A. baumannii is thought to be the most prevalent and virulent member of the genus Acinetobacter, both A. pittii and A. nosocomialis are capable of causing severe human disease and are likely under-represented due largely to technological limitations in species identification across clinical laboratories worldwide [3][4][5].
The ability of Acinetobacter to persist in health care facilities has been an active area of investigation; however, it has been mostly limited to the mechanisms utilized to resist antimicrobial therapy, desiccation, and disinfectants.Little is currently known about the virulence factors employed by Acinetobacter species (spp.) to colonize and infect different human tissues [6][7][8][9].Recent studies have, however, demonstrated that protein glycosylation [10,11], capsule production/modulation [12][13][14], metal acquisition strategies [15,16], outer membrane proteins [17][18][19], and alterations in lipid A [8], all contribute to the ability of medically relevant Acinetobacter species to cause disease.It has also been shown that Acinetobacter spp.produce both type I pili and type IV pili; however, a definitive role for these pili in virulence has not been determined [20][21][22].
Multiple secretion systems have been identified and characterized for their role in the biology and virulence of medically relevant members of the Acb.The most comprehensively studied secretion system in Acinetobacter is the type VI secretion system (T6SS), which has been functionally identified and studied in the medically relevant species A. nosocomialis and A. baumannii, as well as in the non-pathogenic species A. baylyi [23,24].Recently, it was found that several multidrug resistant strains of A. baumannii carry a large, self-transmissible plasmid that encodes for the negative regulators of T6SS.It was found that T6SS is silenced in plasmidcontaining cells while part of the population loses the plasmid and subsequently activates T6SS [25].However, unlike Burkholderia pseudomallei, which utilizes its T6SS to toxically infect eukaryotic cells [26,27], the Acinetobacter T6SS primarily mediates anti-bacterial killing; yet, a recent study identified the Acinetobacter T6SS to be required for full virulence in an insect model [28].A type V system autotransporter, Ata, has also been characterized and found to mediate biofilm formation, adherence to extracellular matrix proteins, as well as virulence in a murine systemic model of Acinetobacter infection [29].Furthermore, plasmid encoded genes required for the biogenesis of a type IV secretion system (T4SS) in A. baumannii and A. lwoffii have been bioinformatically identified [30,31]; however, no empirical evidence demonstrating their function has been presented.To date, no classical toxins have been described nor have any bona fide secretion systems specifically related to disease been discovered in medically relevant Acinetobacter members.
Genes encoding proteins predicted to be associated with a type II secretion system (T2SS) have been identified in A. baumannii [32,33].T2SS are multi-protein complexes, evolutionarily related to type IV pili (T4P) systems, which are responsible for the export of proteins from the periplasmic space to the extracellular milieu or to the outer surface of many Gramnegative bacteria [34,35].The T2SS is composed of 12-15 proteins comprising four subassemblies: a pseudopilus, an inner-membrane platform assembly, an outer-membrane complex, and a secretion ATPase [36].Effector proteins are first translocated to the periplasm by the general secretory (Sec) pathway or the twin arginine transport (Tat) system, where the targeted proteins can then fold into the correct tertiary and/or quaternary structure prior to association with components of the T2SS [37].Competently folded effector proteins can then interact with the different subassemblies of the T2SS and be extruded via interactions with the pseudopilus and the outer-membrane secretin [38].Several Gram-negative pathogens, including Vibrio cholerae [39,40], Legionella pneumophila [41,42], and enterotoxigenic Escherichia coli [43], utilize T2SS for the export of toxins as well as proteins associated with the degradation of biopolymers; thus, T2SS can serve both pathogenic and survival roles for bacteria depending on the environmental niche.
Here, utilizing a proteomics approach coupled with mutational analyses, we demonstrate that Acinetobacter spp.carry a functional T2SS.We also present the type II secretome of A. nosocomialis strain M2.Using a mutational analysis approach, we further demonstrated that both the type IV pili system and the T2SS share a common prepilin peptidase, PilD.Importantly, we show that two of the three identified effectors required chaperones for secretion by the T2SS, one of which is a newly characterized protease/chaperone pair.Lastly, we demonstrated that the Acinetobacter T2SS contributes to the extracellular lipolytic activity, and the virulence in the both the Galleria mellonella infection model and murine pulmonary infection model.

Acinetobacter spp
Previous manuscripts have reported the bioinformatic identification of genes predicted to encode proteins required for the biogenesis of a T2SS in Acinetobacter spp.[32,33].We have also identified homologs of genes associated with the biogenesis of a T2SS in A. nosocomialis strain M2.Here we adopt the gsp nomenclature for general secretory pathway when defining homologous T2SS associated genes in Acinetobacter.Using the Basic Local Alignment Search Tool (BLAST) [44] and homologs of known T2SS-associated genes from V. cholerae, P. aeruginosa, and E. coli, we identified several gsp homologs in all publically available genomes from medically relevant Acinetobacter spp.Unlike many Gram-negative pathogens encoding a T2SS, the genes encoding predicted type II secretion biogenesis proteins were not encoded in a single operon [35], but were grouped into five distinct gene clusters separated over large distances on the chromosome (Fig 1).

Differential secretion of proteins in a gspD-dependent manner
To test the functionality of the T2SS in A. nosocomialis strain M2 we deleted the predicted type II outer membrane secretin gene homolog, gspD, from strain M2.GspD secretin monomers form a dodecamer complex in the outer-membrane that is required for the export of periplasmic effector proteins (Fig 1) [46,47].Using the T2SS deficient M2ΔgspD::kan mutant we probed for differentially secreted proteins by one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).Furthermore, we complemented the gspD::kan mutant and probed for secreted proteins from this genetic background.The secreted protein profiles from all three strains contained an abundance of proteins; however, differences in the The type II secretion associated gene loci as found in A. nosocomialis strain M2.Genes predicted to encode proteins required for the biogenesis of a functioning type II secretion system were clustered into five distinct loci and were distantly spread over the chromosome.A single prepilin/prepseudopilin peptidase homolog was identified, which was located in the previously described pilBCD cluster [45].doi:10.1371/journal.ppat.1005391.g001secreted protein profile from the gspD::kan mutant were clearly evident when compared to the parental strain.At least 4 silver-reactive protein bands were absent in the secreted profile from the gspD::kan mutant when compared to the secreted protein profile from the parental strain (Fig 2A).Importantly, the secreted protein profile from the complemented gspD strain showed the same profile as the parental strain M2 indicating that these differences observed in the secreted protein profile from the gspD::kan mutant were due to the loss of the putative outer membrane secretin and not to the mutational strategy.
2D-DIGE analysis of type II dependent secreted proteins in A. nosocomialis strain M2 Although our 1D SDS-PAGE analysis strongly indicated that A. nosocomialis strain M2 did in fact produce a functional T2SS, the abundance of non-type II secreted proteins would interfere with downstream identification.We therefore proceeded with a two-dimensional difference gel electrophoresis (2D-DIGE) analysis to enhance protein separation.The secreted protein fraction from the wild type strain M2 was compared to the secreted protein fraction from the M2ΔgspD::kan mutant to generate the preliminary type II secretome of A. nosocomialis strain M2 via 2D-DIGE analysis.Analysis of gel images with SameSpots software (TotalLab, New Castle upon Tyne) revealed that 60 spots exhibited a statistically significant average change of at least 4-fold when comparing wild type M2 vs. M2ΔgspD::kan samples.A representative gel image from the 2D-DIGE analysis is shown in Fig 2B .Gel spots were cored using an Ettan Spot Handling Workstation and prepared for in gel trypsin digestion.Peptides were eluted and analyzed using capillary-liquid chromatography-nanospray tandem mass spectrometry.The complete list of proteins identified for each spot as well as a detailed description of the 2D-DIGE analysis and methodologies can be found in S1 Appendix; proteins associated with the largest spot fold change, however, are listed in Fig 2C .Three of the proteins identified in Fig 2C ,  M215_05100, M215_10380, and M215_03235, were of particular interest as all contained domains of known function.The remaining proteins listed in Fig 2C do not contain any known functional domains, with the exception of M215_02250/M215_02255 pair, which was bioinformatically identified as GlyGly-CTERM and rhomobosortase [48].
The top secreted candidate, M215_05100, is an ortholog of the previously identified CpaA metallopeptidase from the M72 family of peptidases, which was proposed to cleave both factor V and fibrinogen [49].The M72 peptidases are characterized as peptidyl-Asp-endopeptidases containing the HEXXHXXGXX active site, where a zinc ion is predicted to be bound by three histidine residues, and the glutamate is predicted to be the catalytic residue [50].The M215_10380 locus encodes an ortholog of the previously characterized LipA lipase from A. baylyi [51,52], which contains an alpha/beta hydrolase fold from the homologous family abH15.02(B.cepacia lipase-like) within the abH15 superfamily (Burkholderia lipase superfamily) as determined by the Lipase Engineering Database [53].These lipases are predicted to have a catalytic triad of a serine, a glutamate or aspartate, and a histidine.Lastly, the M215_03235 locus encodes for another protein containing an alpha/beta hydrolase fold; however, the M215_03235 gene product does not have homology to any know lipases within the Lipase Engineering Database and has yet to be characterized in Acinetobacter.
The prepilin peptidase, PilD, is also the pre-pseudopilin peptidase of the Acinetobacter T2SS BLAST analysis revealed the presence of only a single prepilin peptidase, gspO/pilD, which was previously designated PilD and reported to be the major prepilin peptidase for the type IV pili (T4P) system in Acinetobacter (Fig 1) [45].Given that only one gspO/pilD homolog was A representative gel image showing Cy3 (ΔgspD::kan, green) and Cy5-labeled (parent, red) proteins that were isoelectric focused on pH strips (3-10), separated by size using SDS-PAGE, and visualized using a Typhoon 9400 variable mode imager.A merged image of the Cy3 and Cy5-labeled proteins is shown.Proteins with greater abundance in the ΔgspD::kan sample appear green and proteins with greater abundance in the parent strain sample appear red.Proteins that did not change relative abundance between the two samples appear yellow.(C) Putative T2S-dependent proteins identified via 2D-DIGE analyses.Protein candidates associated with the largest spot fold change were bioinformatically examined for the presence of a signal peptide and putative functions. 1The protein score is derived from Mascot and provides an indication of how well the peptides matched the indicated protein sequence.The actual score is calculated by the following equation: protein score = -10*Log(P), where P is the probability that the protein match is a random event.Scores above 100 indicate that p < 0.05. 2 The protein match score indicates the number of unique peptides that matched the sequence of the identified protein.Two unique peptide matches to a protein sequence confirms the identity of a protein.identified in strain M2's genome as well as in A. baumannii ATCC 17978 and 19606, we hypothesized that the previously identified prepilin peptidase, PilD, was also the pre-pseudopilin peptidase required for the T2SS.To this end, we cloned and heterologously expressed the predicted major pseudopilin, gspG, with a carboxy-terminal FLAG tag in the wild type M2 background, the ΔpilD::kan mutant, and its respective complement in order to probe for pseudopilin processing.As expected GspG-FLAG expression was detected in all three backgrounds; however, GspG-FLAG from both the wild type M2 and the complemented pilD strain migrated with an increased electrophoretic mobility as compared to GspG-FLAG from the ΔpilD::kan strain (Fig 3).The increase in electrophoretic mobility was most likely due to the loss of the leader sequence of GspG; furthermore, PilD was required for the processing observed.Lastly, an additional band of intermediate electrophoretic mobility was detected only in the ΔpilD:: kan background.We hypothesize this form of GspG-FLAG to be a degradation product.
The type II secretion of the CpaA metallopeptidase is dependent on a novel protease chaperone, CpaB Our 2D DIGE analysis indicated that the CpaA metallopeptidase was secreted via the T2SS; therefore, we used an immunoblotting approach to verify CpaA secretion was type II dependent.We cloned the cpaA gene with its predicted native promoter into the Acinetobacter-E.coli shuttle vector pWH1266 [54], containing a hexa-histidine tag onto the carboxy terminus of cpaA.Hexa-histidine tagged CpaA was expressed in trans in multiple genetic backgrounds to probe for expression and secretion.CpaA-His expression was detected in all strains tested, however, it was only detected in the secreted fractions from strains predicted to have a fully functioning T2SS (Fig 4C).Specifically, neither the ΔgspD::kan mutant nor the ΔpilD::kan mutant secreted CpaA-His, indicating the dependency of the T2SS for active export of CpaA.All strains and fractions were also analyzed for RNA polymerase expression, which served as a lysis control.CpaA-His expression was detected in all strains carrying pWH-cpaA-his; however, CpaA-His secretion was only detected in strains predicted to produce a functioning T2SS.(D) Western blot analysis on whole cell lysates and secreted protein fractions probing for CpaA-His from the parent strain, the ΔcpaB::frt mutant, and the cpaB complemented strain.CpaA-His was detected in all strains; however, CpaA-His secretion was not detected in the As expected secretion was independent of the type IV pilus as the ΔpilA::frt mutant displayed active secretion of CpaA-His.
Immediately downstream of cpaA is the M215_05105 open reading frame, which when analyzed by BLASTp did not identify any known functional domains.However, when the M215_05105 ORF was analyzed by Domain Enhanced Lookup Time Accelerated (DELTA) BLASTp, which has higher sensitivity than BLASTp [55], the M215_05105 ORF was found to contain a domain from the SRPBCC superfamily (Fig 4A and 4B).Proteins carrying a domain from the SRPBCC superfamily are predicted to contain a deep hydrophobic ligand-binding pocket and have chaperone-like activity [56].We thus hypothesized that the M215_05105 gene product, designated CpaB due to its proximity to CpaA, was a CpaA-specific chaperone.To test our hypothesis, we deleted the cpaB gene and probed for CpaA-His expression and secretion.As shown in Fig 4D, CpaA-His expression was detected in the ΔcpaB::frt mutant; however, CpaA-His was not secreted, indicating that CpaB was required for CpaA secretion.Importantly, we were able to reintroduce the cpaB allele and restore the active secretion of CpaA-His.
To further demonstrate the dependency of CpaA secretion on CpaB, we heterologously expressed cpaA-his alone or in tandem with cpaB in A. baumannii ATCC 19606, which does not encode for orthologs of either the CpaA metallopeptidase or the CpaB chaperone, yet is predicted to produce a functional T2SS.As shown in Fig 4E, CpaA-His was expressed but not secreted by 19606 cells when the pWH-cpaA-his plasmid was introduced, however, when both cpaA-his and cpaB were co-expressed, CpaA-His was secreted, indicating that CpaA secretion is not only dependent on a functional T2SS, but also on the chaperone activity of CpaB.
The LipA lipase is exported by the type II secretion system, is lipolytic towards neutral triglycerides, and is dependent on the LipB chaperone for secretion The M215_10380 ORF, encoding for a LipA ortholog, was also identified in our 2D-DIGE analysis as a type II effector.It has been previously demonstrated in A. baylyi and Pseudomonas that secretion and over-expression of LipA orthologs are dependent on a LipB-like chaperone [51,57].In A. nosocomialis M2, a lipB homolog is adjacent to lipA (Fig 5A and 5B).When LipA-His was over-expressed from the pWH-lipA-his plasmid, we did not detect its secretion.However, when we co-expressed the upstream lipB gene with lipA-his, LipA-His was expressed and secreted in all backgrounds predicted to have a functional T2SS (Fig 5C).LipA-His was neither detected in the secreted fraction from the ΔgspD::kan mutant nor the ΔpilD::kan mutant.Secretion was also independent of the type IV pilus fiber itself (Fig 5D).We also confirmed that LipA was secreted in a LipB chaperone-dependent manner by A. baumannii To confirm that LipA is in fact a lipase, we purified culture supernatants from multiple genetic backgrounds and probed for lipolytic activity as determined by a modified para-nitrophenol palmitate (p-NPP) assay [58].As seen in Fig 5F, culture supernatants from the wild type M2 exhibited lipolytic activity as demonstrated by an increase in the absorbance at 410nm (A 410 ) over a 12-hour time period.Culture supernatants from the ΔgspD::kan mutant displayed only minimal increases in the A 410 indicating almost a complete lack of lipase activity.Importantly, the complemented gspD strain displayed very similar increases in the A 410 when compared to the wild type, indicating that the lipase activity in culture supernatants was mainly dependent on the T2SS.Culture supernatants from the lipA::kan mutant exhibited an approximately 50% reduction in lipase activity; furthermore, the complemented lipA strain regained activity; in fact, culture supernatants from the complemented lipA strain displayed approximately a 30% increase in lipase activity over the wild type strain.Next we purified culture supernatants from the lipB::frt mutant and found that it displayed the same profile as the lipA mutant when measuring the A 410 ; however, when we reintroduced the lipB gene into the lipB:: frt mutant, we observed minimal complementation (Fig 5F).
The M215_03235 locus encodes for a newly characterized lipase, LipH, which is also secreted in a type II dependent manner The 2D-DIGE analysis revealed that the spot corresponding with the M215_03235 protein was associated with an 8.1 fold change when compared to the ΔgspD::kan mutant.The M215_03235 gene encodes for a protein containing multiple predicted domains including a LIP domain (pfam03583), a DAP2 domain (COG1506), and two AB hydrolase_5 domains (pfam 12695).Given that all of these domains are associated with predicted lipase/esterase activity, we have designated M215_03235 as lipH in order to avoid confusion with previously characterized lipases.
To confirm that LipH was secreted in a T2SS-dependent manner, we utilized a similar approach as described above where we cloned and tagged LipH into pWH1266 with a carboxyterminal his tag.We then introduced this construct into multiple strains and probed from LipH-His expression and secretion.As seen in Fig 6A, LipH-His was detected in whole cell lysates of all strains tested; however, LipH-His was found to only be secreted in strains predicted to express a functional T2SS.We further assessed the ability of a panel of clinical isolates to secrete LipH-His.As shown in Fig 6B, LipH-His expression and secretion was detected in all clinical isolates tested.
Because alpha/beta hydrolase domains, such as the one present in LipH, are commonly found in lipases, we verified that LipH has lipolytic activity.We constructed a ΔlipH::kan mutant as well as a lipH complemented strain and subjected these strains to the p-NPP assay utilized above for LipA.As seen in Fig 6C, the ΔlipH::kan mutant displayed an increase in the A 410 , indicating lipolytic activity; however, the increase was substantially lower than both the parent strain as well as the lipH complemented strain indicating that the LipH protein is a lipase.
The Acinetobacter T2SS is required for optimal virulence in the Galleria mellonella model of infection The greater wax moth, Galleria mellonella, has been routinely used to assess the virulence of Acinetobacter [59].Furthermore, strains with attenuated virulence in the G. mellonella model (C) Western blot analysis on whole cell lysates and secreted protein fractions probing for LipA-His.All strains and fractions were also analyzed for RNA polymerase expression, which served as a lysis control.LipA-His expression was detected in all strains carrying pWH-lipA-his as well as pWH-lipB-lipA-his; however, LipA-His secretion was only detected in strains co-expressing the chaperone LipB as well as a predicted functioning T2SS.(D) Secretion of LipA-His was independent of the type IV pilus, as indicated by active secretion in the ΔpilA::frt mutant.(E) Western blot analysis on whole cell lysates or secreted protein fractions from A. baumannii ATCC 19606 carrying the empty vector, the pWH-lipA-his vector, or the pWH-lipB-lipA-his vector.LipA-His expression was detected; however, LipA-His secretion was only detected when the lipB gene was co-expressed with lipA-His.(F) Lipolytic activity of purified culture supernatants from the indicated strains as determined by a modified p-NPP assay.Increases in the A 410 indicate lipolytic activity.A 410 measurements were recorded every 30 minutes for 12 hours.Three biological replicates with three technical replicates were used for analysis.doi:10.1371/journal.ppat.1005391.g005Fig 6 .The newly characterized LipH is secreted by the T2SS and displays lipase activity.(A) Western blot analysis on whole cell lysates and secreted protein fractions probing for LipH-His.All strains and fractions were also analyzed for RNA polymerase expression, which served as a lysis control.LipH-His expression was detected in all strains carrying the pWH-lipH-his; however, LipH-His secretion was only detected in strains predicted to produce a functioning T2SS.(B) Western blot analysis on whole cell lysates and secreted protein fractions from a range of Acinetobacter clinical isolates heterologously have also been shown to have attenuated virulence in murine models of infection [60].In order to assess the role of the Acinetobacter T2SS in the G. mellonella model, we first determined the LD 50 for the wild type A. nosocomialis strain M2.Groups of ten larvae were each injected with 10μL of either approximately 10 5 , 10 6 , or 10 7 total CFU of strain M2, incubated at 37°C for 24 hours, and checked for viability as determined by accumulation of melanin and loss of movement.From these studies, the LD 50 was determined to be approximately 3X10 6  The T2SS of A. nosocomialis strain M2 is required for optimal virulence in the G. mellonella infection model.(A) Groups of G. mellonella larvae were injected with 10μL of either the parent strain, the ΔgspD::kan mutant, or the complemented gspD::kan strain at an inoculum previously determined to be the equivalent of the LD 50 for the parent strain.Larvae were checked for viability as determined by melanin accumulation and motility.(B) Groups of G. mellonella were injected with 10μL of either the parent strain, the ΔgspD::kan mutant, or the complemented gspD::kan strain at an inoculum previously determined to be the LD 50 for the ΔgspD::kan mutant.Larvae were checked for viability as determined by melanin accumulation and motility.Survival curves were determined to be statistically significant using the Mantel-Cox test (P = 0.0147).doi:10.1371/journal.ppat.1005391.g007strain of A. nosocomialis with an unmarked, in-frame deletion of gspD, which encodes for the predicted outer-membrane secretin.Prior to infection studies, we verified that the newly generated M2ΔgspD::frt mutant was in fact impaired in secretion of type II effector proteins (S3 Fig).
Using our previously described murine infection model [62], we performed infection experiments with either the wild type A. nosocomialis strain M2, the unmarked, isogenic M2ΔgspD:: frt mutant, or its respective gspD complemented strain.Mice were intranasally inoculated with 1X10 9 CFU, as we previously determined that inoculating mice with this dose of wild type bacteria resulted in full murine viability, yet, resulted in significant organ-specific bacterial burden (S4 Fig) .Groups of mice were individually administered an intransal inoculation of either the wild type strain, the ΔgspD::frt mutant, or the respective complemented gspD strain.Thirty-six hours post-infection, mice were sacrificed and the lungs, spleen, and livers were harvested in order to determine total bacterial burdens.As seen in Fig 8A, mice infected with either the wild type strain or the complemented gspD strain all had high bacterial burdens in the lungs.Furthermore, bacterial burdens displayed limited variability indicating a full level of complementation for the gspD complementation strain.Mice infected with the ΔgspD::frt mutant displayed significantly lower bacterial burdens in the lung when compared to either the wild type or complemented gspD strain.A similar trend was also observed for bacterial burdens in the spleen, where, mice infected with either the wild type or the complemented gspD strain had significantly higher bacterial burdens (Fig 8B).We also enumerated bacterial colony forming units from the livers of infected mice and did not observe any significant differences between the cohorts (Fig 8C ).

LipB and CpaB belong to a distinct class of membrane-bound T2SS chaperones found in Gram-negative bacteria
We have shown that two of the three secreted type II effectors identified in A. nosocomialis strain M2 require specific chaperones for secretion.To date only the lipase-specific foldases (Lifs) have been characterized as chaperones for type II effectors [51,[63][64][65].Indeed, a complex A. nosocomialis strain M2 required its T2SS for optimal colonization of both the lungs and spleen.Mice were intranasally inoculated with 1X10 9 CFU of either the wild type, ΔgspD::frt mutant, or the gspD complemented strain.After 36h post-infection, mice were sacrificed, organs harvested, and CFUs enumerated from homogenized tissue.(A) Total CFUs from the lung demonstrated a statistically significant difference between the complemented gspD strain and the ΔgspD::frt mutant (Kruskal-Wallis non-parametric with Dunn's multiple comparison test, ANOVA, *p = 0.0467).(B) Total CFUs from the spleen demonstrated statistically significance comparisons for both comparisons of either the wild type and the ΔgspD::frt mutant as well as the complemented gspD strain and the ΔgspD::frt mutant (One way ANOVA, **p = 0.0230, ***p = 0.0078).Open boxes indicate CFUs that were below the limit of detection.(C) Total CFUs form the liver did not demonstrate any statistically significant differences.Open boxes indicate CFUs that were below the limit of detection doi:10.1371/journal.ppat.1005391.g008 of the B. glumae LipA/Lif has been crystallized [66].The Lifs are unique steric chaperones, which have an N-terminal membrane-anchor and a C-terminal domain that facilitates proper folding of their cognate lipase upon entry into the periplasm [67].Furthermore, the first characterization of a chaperone participating in the secretion of a type II secreted protein from Acinetobacter was described in 1995.These authors demonstrated that a lipase specific chaperone, designated LipB, was required for secretion of the LipA lipase.They found that the C-terminal domain of the LipB chaperone was located outside of the cytoplasm.Lastly, in contrast to what had been previously found in Pseudomonas strains, the authors found that lipB was actually encoded upstream of lipA [51,52].We have expanded upon this paradigm with the identification of a novel protease/chaperone pair (CpaA/B).Furthermore, we hypothesized this phenomenon to be more widespread.In order to identify putative chaperones of type II secreted proteins, we first searched for open reading frames (ORFs) encoded adjacently to known type II effectors that were predicted to be part of the same operon.We then narrowed our search to ORFs that encode for proteins with a predicted N-terminal transmembrane domain as this feature is shared both by the Lifs and the newly characterized CpaB chaperone.As found in Table 1, we were able to identify several putative chaperones of type II effectors in diverse Gram-negative bacteria such as V. cholerae, P. aeruginosa, and B. pseudomallei, which suggests that CpaB, LipB, and Lifs belong to a family of membrane-bound chaperones involved in T2SS secretion.

Discussion
Acinetobacter spp.have rapidly emerged as significant opportunistic pathogens afflicting healthcare facilities worldwide.Although sophisticated studies track the epidemiology of Table 1.Known and putative type II chaperones.Open reading frames of unknown function within the same operon as known type II effectors were analyzed for N-terminal transmembrane helices.Transmembrane domains were predicted using the TMHMM Server v. 2.0.*The lipB annotation in P. aeruginosa PAO1 is truncated, excluding the N-terminal sequence.Here we have included the putative full length open reading frame for analysis.outbreaks worldwide, our collective understanding of the molecular mechanisms employed by Acinetobacter spp. to cause disease is in its infancy.In this work, we combined bioinformatics, proteomics, mutational analyses, and virulence assays to demonstrate that Acinetobacter spp.produce a functional T2SS, which is required for the secretion of multiple proteins that are required for full virulence.Importantly, this is the first bona fide secretion system required for virulence in a mammalian model identified in Acinetobacter.Notably, two of the three secreted proteins characterized in this study require dedicated chaperones for type II secretion.While this paper was under revision, an article reporting the presence of a functioning T2SS in A. baumannii ATCC 17978 was published [76].In this work, it was found that 17978 also required a T2SS for the secretion of the LipA lipase and growth on minimal media with olive oil as the sole carbon source.It was also found that both the 17978ΔgspD and 17978ΔlipA mutants were less fit in a murine septicemia model when competed against the parental strain.

Chaperone/ PutativeChaperone
Typically, T2SSs secrete as many as 18-25 proteins and facilitate the delivery of major virulence factors to the extracellular environment for many important human pathogens, such as Legionella pneumophila and V. cholerae [40,42].Here, we utilized the 2D-DIGE method coupled with mutational analyses to characterize the type II secretome for A. nosocomialis strain M2.Our analysis identified over 60 spots with a 4-fold difference when comparing the wild type M2 vs. M2ΔgspD::kan mutant; however, we concentrated our efforts on three proteins that contain domains of known functions.Other studies will be needed to determine the role of the remaining type 2 effector candidates and of individual secreted proteins in Acinetobacter pathobiology given the importance of this system in virulence.
The genetic architecture of T2SSs usually consists of between 12 and 15 genes, most of which appear to be organized in a single operon [35].From a regulatory standpoint, the single operon arrangement of T2SS associated genes would seem to be the simplest to transcriptional control.However, as noted above, the T2SS associated genes from A. nosocomialis strain M2 are found in five distinct genetic loci, a genetic arrangement that resembles the type IVa pilus system [77].Furthermore, this dispersed genetic arrangement is highly conserved across different Acinetobacter species, including the pathogenic species A. baumannii and the non-pathogenic species A. baylyi.Closer examination of each T2SS gene cluster does not provide any obvious insights into the regulatory mechanisms as some T2SS genes appear to be in putative operons with other genes not known to be associated with T2SSs.Outside of the genus Acinetobacter the same genetic architecture can also be found in bacteria from the genus Psychrobacter.
As demonstrated previously, the prepilin peptidase PilD was required for major pilin processing and proper functionality of T4P in A. nosocomialis strain M2 [45].Our current data demonstrated that PilD is also required processing of the predicted major pseudopilin, GspG, and thus secretion of T2S substrates.Given the strong evolutionary relatedness between the T4P system and the T2SS, the phenomenon of sharing protein components between two functionally distinct systems does not seem impractical, nevertheless, it is uncommon.To date only D. nodosus [78], P. aeruginosa [79], V. cholerae [80], and L. pneumophila [81,82] have been demonstrated to share a prepilin peptidase between both the T4P system and a T2SS.
Of the three type II effectors studied, only LipA has previously characterized orthologs, which were primarily described in Pseudomonas and also require a chaperone [83].However, to date, none of these lipases have been connected to pathogenesis.We demonstrated that the LipA lipase was responsible for approximately half of the lipase activity observed from the secreted fraction of the wild type strain M2.As expected, LipA activity was also dependent on the LipB chaperone, as supernatants from the ΔlipB::frt mutant displayed nearly identical lipase activity levels as the ΔlipA::kan mutation.However, our lipB complemented strain only marginally increased the lipase activity of the ΔlipB::frt mutant, indicating that even though we constructed an in-frame, unmarked mutation in the gene, we may still be observing polar effects on lipA transcription.The lipA gene is 81bp downstream of the lipB gene and therefore could potentially have its own promoter that is partially contained within the 3' region of the lipB gene.We and others have observed similar cryptic promoter events during previous studies of the pilTU gene cluster, where an in-frame, unmarked mutation of pilT still had polar effects on pilU expression [45,84].
Even in the absence of lipA, culture supernatants retained residual lipase activity as compared to the gspD mutant strain.As such, we found that LipH mediated lipase activity of culture supernatants as well.A BLASTp search of LipH orthologs outside of Acinetobacter identified similar proteins found in bacteria from the genus Myriodes, some of which act as opportunistic human pathogens [85], as well as bacteria from the genus Bacillus; however, none of those orthologs have been characterized.
Using A. nosocomialis strain M2 as our model system we demonstrated that LipH secretion was indeed dependent on a functional T2SS.We also demonstrated that T2SS is conserved and functional across Acinetobacter spp.via immunoblotting of epitope tagged effectors.Specifically, we showed that LipH from M2 was secreted by a panel of Acinetobacter clinical isolates, including, A. calcoaceticus, A. baumannii, A. pittii, and A. junnii.We also demonstrated that A. baumannii ATCC 19606 could secrete both LipA and CpaA; however, as expected the respective chaperones for each protein were required for active secretion.These data strongly suggest the presence of a functional T2SS in the majority of medically relevant Acinetobacter spp.This hypothesis is further supported by the fact that genes predicted to encode proteins required for the biogenesis of the T2SS are highly conserved and distributed amongst Acinetobacter spp.
The remaining effector characterized in our study was the CpaA metallopeptidase.CpaA was previously purified from culture supernatants [49]; however, its mechanism of secretion was not determined.It was previously shown that CpaA is involved in degradation of Factor V and fibrinogen, which would result in a decrease in clotting activity.Here, we demonstrated that CpaA was secreted in abundance in a type II dependent manner, yet, was also dependent on a novel chaperone, designated CpaB.CpaB is the first characterized T2SS chaperone devoted to the secretion of a protease.Topological modeling of the CpaB chaperone predicts a single N-terminal transmembrane domain with the majority of the protein exposed to the periplasm [86,87].The periplasmic exposed C-terminal domain of CpaB was predicted by DELTA BLASTp to contain a domain from the SRPBCC superfamily present in the co-chaperone eukaryotic protein Aha1, the activator of Hsp90 complex [56].The SRPBCC domains are predicted to have deep hydrophobic ligand binding pockets.A BLASTp search of CpaB orthologs outside of Acinetobacter only identified two weak orthologs from Lysobacter antibioticus; however, a DELTA BLASTp search for CpaB orthologs outside of Acinetobacter primarily identified Aha1 as the closet ortholog, suggesting a possible eukaryotic ancestry.Currently, we hypothesize that the CpaA metallopeptidase is trafficked through the Sec system, as is the case for most type II secreted substrates.There, CpaA can interact with CpaB as CpaB is predicted to contain a single transmembrane domain with the majority of the protein exposed to the periplasmic space.Upon entry into the periplasmic space of CpaA from the Sec system, CpaB could facilitate proper folding of CpaA due to the requirement of type II secretion systems for competently folded proteins for active secretion.
The potential role of the CpaA metallopeptidase in Acinetobacter pathogenesis and evolution is quite intriguing.Firstly, the type strains A. baumannii ATCC 17978 and 19606, two of the more primitive Acinetobacter spp.used as model organisms do not contain orthologs of the CpaAB system, indicating a horizontal acquisition event within the last 70 years.Analysis of the GC content of the cpaAB locus and the surrounding DNA support this hypothesis.It is tempting to speculate, that given the predicted recent acquisition of the CpaAB protease/ chaperone system and the role of the T2SS in Acinetobacter virulence, CpaA may one of the major virulence factors of some pathogenic Acinetobacter spp.Future work will be aimed at deciphering the role of CpaA in the virulence assays utilized within this study.
As mentioned above, LipB and CpaB act as specific chaperones for LipA and CpaA respectively.Some effectors secreted via a type III secretion system (T3SS) also require specific chaperones that have collectively been named "T3SS chaperones" [88].T3SS chaperones do not present sequence similarity, but they are easily identified because they are encoded next to their cognate effector and most of them contain similar molecular weight and isoelectric points.Similarly, we define a "T2SS chaperone" as a protein encoded adjacently and co-regulated with a type II effector, that contains both an N-terminal transmembrane domain, and an exposed C-terminal region to the periplasm, and that is required for secretion of the cognate effectors.We identified "type II chaperones" in multiple Gram-negative species.Interestingly, LipB from Pseudomonas aeruginosa is a previously characterized chaperone that serves two T2SS effectors, LipA which is encoded next to LipB as well as LipC, which is encoded more than 2 Mb away [72].This indicates that the T2SS chaperones family may be more widespread than we propose here.
We determined that the Acinetobacter T2SS was required for virulence.We first determined that the mutants unable to produce a functioning T2SS were attenuated in the G. mellonella infection model.Given the high level of concordance between mutants attenuated in the G. mellonella model and mammalian models, we hypothesized a more relevant in vivo role for the Acinetobacter T2SS.We thus choose to investigate the role of T2S in a murine pulmonary infection model.Specifically, we observed high CFUs for the wild-type strain in the lungs after 36h infection period and also observed dissemination to both the liver and spleen.Using an unmarked, in-frame deletion of gspD strain and its respective complemented strain, we were able to demonstrate that the T2SS was indeed required for optimal colonization of both the lungs and spleen, but not the liver.Remarkably, we observed almost a two log decrease in CFUs in the lungs and spleen of mice infected with the gspD mutant strain when compared to either the wild type or the complemented strain.Many studies focusing on Acinetobacter pathobiology have utilized a similar murine pneumonia model of infection and also observed differences of around 2 logs; however, these mutants had defects in two-component regulatory systems, metabolism, and/or stress responses, all of which could have more pronounced global effects on Acinetobacter biology that mediate defects in colonization [32,89].
Herein, we have provided evidence of both a functional T2SS in many Acinetobacter spp. as well as demonstrated its importance in Acinetobacter pathogenicity.However, the exact role for each T2S effector proteins in Acinetobacter pathogenicity has yet to be determined.As such we plan to next probe the role of specific effectors in mediating the colonization phenotypes observed, with an emphasis on the most highly secreted protein, the CpaA metallopeptidase.Furthermore, our study highlights the use of other clinically relevant members of the genus Acinetobacter outside of A. baumannii in order to gain insights into the pathogenesis of clinically relevant Acb members.Although type strains like A. baumannii ATCC 17978 and 19606 have served well as model strains for Acinetobacter pathogenicity, their relative old age makes them less representative of current epidemic strains, which contain more antibiotic resistance cassettes and possibly novel virulence attributes.

Strains, plasmids, and growth conditions
Bacterial strains and plasmids utilized within this study can be located in the S1 Table .All bacterial strains were grown on L-agar at 37°C.Antibiotic selection for E. coli strains was used at the following concentrations: 100μg ampicillin/mL, 5μg tetracycline/mL, and 20μg mL.Antibiotic selection for Acinetobacter strains was used at the following concentrations: 200μg ampicillin/mL, 5μg tetracycline/mL, 20μg kanamycin/mL, 12.5μg chloramphenicol/mL.Sucrose was used at a final concentration of 10% for counter selecting Acinetobacter strains that lost the sacB cassette.

Generation of bacterial mutants and complemented mutants
All marked and unmarked mutants were generated using the previously published methodologies found in [22,45] using the In-Fusion HD EcoDry cloning kit.The In-Fusion HD EcoDry cloning kit was used to generate the interrupted gene constructs as described in the supplemental material of [22] and introduced into strain M2 via natural transformation as described in [45].For strains containing the kan-sacB cassette, a tri-parental mating strategy was used to transiently introduce the pFLP2 plasmid as described in [23], in order to replace the cassette with an frt scar.Strains designated with the "::frt" nomenclature contain a frt scar in place of the target gene.Each mutation was complemented using the mTn7 described in [22].A complete list of primers for mutational analyses can be found in S2 Table.

Bioinformatic analysis
The Basic Local Alignment Search Tool (BLAST) tool was utilized in order to identify known gene homologs of type II secretion system related genes in Acinetobacter.

One dimensional SDS-PAGE analysis of secreted proteins
Fifty milliliter cultures of each strain was grown for 18 h in M9 salts supplemented with 1% casamino acids and 1% glucose with 180 rpm.The secreted proteins were separated from the whole cells by centrifugation at 4000rpm for 10 mins.The supernatants were then further purified by filtration through 0.22 micron filters.The secreted proteins were then concentrated to ~100μL using Amicon Ultra Centrifugal Filter units with a 10kDa cutoff.Laemmli buffer with β-mercaptoethanol was added to each fraction and the samples were heated to 100°C by boiling in water for 10 mins.Twenty microliters of each sample was then separated by SDS-PAGE in a 4-20% gradient gel and subsequently silver stained.

2D-DIGE analyses
Secreted proteins used for the 2D-DIGE analysis were prepared as described in the above section discussing 1D SDS-PAGE analysis of secreted proteins for both the wild type A. nosocomialis strain M2 and its isogenic gspD::kan mutant.A detailed protocol for the 2D-DIGE analysis can be located in S1 Appendix.All 2D-DIGE experiments were performed by the Campus Chemical Instrument Center Mass Spectrometry and Proteomics Facility at The Ohio State University.

Generation of pWH1266 carrying effectors and effector/chaperone pairs
In order to validate the 2D-DIGE analysis identifying the putative type II secreted proteins of strain M2, selected effectors and effector/chaperone pairs were cloned into the Acinetobacter-E.coli shuttle vector pWH1266.Briefly, lipA, cpaA, lipH, lipBA, and cpaAB loci were PCR amplified using the primers listed in S2 Table using strain M2 genomic DNA as template for PCRs.Each PCR product was purified, digested with PvuI-HF, and ligated into pWH1266 that was predigested with PvuI-HF and treated with phosphatase.The ligations were transformed into E. coli TOP10 cells and transformants were selected for on L-agar supplemented with tetracycline.Transformants were sub-cultured and each plasmid was purified and verified sequencing.The carboxy-terminal His tag was added to lipA, lipH, and cpaA with a second PCR, where the respective forward primer included a 5' overhang encoding for the His-tag using with the primers listed in S2 Table .The PCR products were purified, DpnI treated, and self-ligated.The ligations were transformed into TOP10 cells and transformants were selected on L-agar supplemented with tetracycline.Transformants were sub-cultured and the plasmids were purified and verified by sequencing.Vectors expressing the His-tagged constructs were electroporated into electrocompetent Acinetobacter spp.and transformants were selected for on L-agar supplemented with tetracycline.

Generation of pWH-gspG-FLAG
To test for PilD-dependent processing of GspG, the gspFG locus including the predicted native promoter was PCR amplified using the primers listed in S2 Table .The PCR product was purified, digested with PvuI-HF, and ligated into pWH1266 that was predigested with PvuI-HF and treated with phosphatase.The ligations were transformed into TOP10 cells and transformants were selected on L-agar supplemented with tetracycline.Transformants were sub-cultured and the plasmids were purified and verified by sequencing.To remove the gspF gene, an inverse PCR strategy was employed to PCR out gspF leaving the ATG start codon and the last 21 bp in order to generate an in-frame deletion.The PCR product was purified, treated with kinase, and self-ligated.The ligations were transformed into TOP10 cells and transformants were selected on L-agar supplemented with tetracycline.Transformants were sub-cultured and the plasmids were purified and verified by sequencing.The FLAG tag was PCR amplified onto the carboxy terminus of gspG as described above using the primers listed in S2 Table .The PCR product was purified, treated with kinase, and self-ligated.The ligations were transformed into TOP10 cells and transformants were selected on L-agar supplemented with tetracycline.Transformants were sub-cultured and the plasmids were purified and verified by sequencing.The pWH-gspG-FLAG construct was then electroporated into electrocompetent A. nosocomialis strains.

Type II secreted protein detection
Strains carrying His-tagged lipA, lipH, or cpaA were screened for active secretion via immunoblotting.Briefly, strains were struck and grown overnight on L-agar supplemented with tetracycline at 37°C.Bacteria were swabbed from the plate, resuspended in LB broth, and used to inoculate 10mL of LB broth to an OD 600 of 0.05 supplemented with tetracycline.The cultures were grown to mid-log phase, normalized to an OD 600 of 0.5, then processed for whole cell fractions and secreted fractions.Whole cell fractions were obtained by removing 1mL of the normalized mid-log cells, pelleting the cells by centrifugation, and removing the supernatant.Bacterial pellets were then resuspended in 50μL of 1X Laemmli buffer.Secreted fractions were obtained by pelleting the normalized mid-log cultures by centrifugation and carefully removing 1mL of the supernatant.Secreted proteins were precipitated by the addition of 250μL of a saturated trichloroacetic acid solution.Precipitated proteins were incubated on ice for 10-30 mins, pelleted by centrifugation, and washed twice with ice-cold acetone.Residual acetone was removed by heating the samples at 95°C.Precipitated proteins were resuspended in 100μL of 1X Laemmli buffer.Both whole cell fractions and secreted fractions were boiled in 1X Laemmli buffer for 10 mins and subsequently used for immunoblotting.Proteins were separated on a 10% SDS-PAGE gel, transferred to nitrocellulose, and probed for RNA polymerase and 6X-Histidine tagged proteins according to our previously published methodologies.

Lipase assay
In order to determine lipolytic activity of secreted protein a modified version of the para-nitrophenol palmitate (p-NPP) lipase assay was performed.Secreted protein fractions were purified from select strains as described above with slight modifications.Briefly, 2.5mL of culture supernatant was clarified by centrifugation and then filtered through 0.22μM PVDF filters.The secreted proteins were buffer exchanged into 50mM Tris and were concentrated to ~250μL using Amicon Ultra Centrifugal Filter units with a 10kDa cutoff and promptly used for the lipase assay.Lipase activity was determined by measuring the absorbance at 410nm at 37°C using p-NPP as a substrate.The p-NPP solution was freshly prepared for each assay by diluting solution A (0.1g p-NPP in 100mL isopropanol) 1:10 with solution B (1g gum Arabic, 2g sodium deoxycholate, 5mL triton X-100, 50mM Tris-HCl pH 8 in 900mL).Seventy microliters of the p-NPP solution was then added to 30μL of the concentrated, clarified secreted protein fractions from a respective strain.Kinetic measurements recording the absorbance at 410nm were then performed over the designated time frame at 37°C with orbital shaking between each absorbance reading.Absorbance measurements were captured using the Synergy HTX multi-mode reader from BioTek.Each experiment was performed in triplicate with three technical replicates per sample.

Galleria mellonella infection
A. nosocomialis M2, the ΔgspD::kan mutant and the complemented strain were grown in LB broth overnight in an orbital shaker (37°C, 200rpm).The overnight cultures were diluted to a starting OD 600 0.05 and grown at 37°C with 200rpm to a final OD 600 of 0.5.0.5ODs was pelleted by centrifugation, washed with filter sterilized PBS and resuspended at and OD of 0.5/ml, 0.158OD/mL and 0.05OD/mL in filter sterile PBS.The CFU/mL at 0.5OD/mL was determined to be 10 9 .Serial dilution of the 0.5OD/mL sample was performed.Larvae were injected with 10uL of sterile PBS, 10 6 or 10 7 CFU. 3 groups of 10 larvae were injected per experimental group.The larvae were scored as live/dead depending on their response to physical stimulus approximately every 5 hours.The number of bacterial cells injected into the larvae was determined by plating 10-fold serial dilutions on LB agar and performing CFU counts after overnight incubation at 37°C.

Mouse model of pneumonia
All infection experiments were approved by the Vanderbilt University Institutional Animal Care and Use Committee.Wild-type C57BL/6 mice, obtained from Jackson Laboratories, were used for single infection experiments with either the wild type A. nosocomialis M2, the M2ΔgspD::frt mutant, or the respective gspD complemented strain.Overnight cultures of each strain were sub-cultured 1/1000 into 50 mL LB broth and grown with shaking at 37°C in 250-mL flasks.Bacterial cells were harvested by centrifugation during logarithmic growth, washed twice with phosphate buffered saline (PBS), and suspended in PBS.Nine-week old male mice were inoculated intranasally with a total of 7-8 X 10 8 cfu in 30 μL.At 36 h postinfection, mice were euthanized and CFUs were enumerated from the lungs, livers, and spleens following tissue homogenization and dilution plating to LB agar medium.The data were log transformed and analyzed for Gaussian distribution using the D'Angostino-Pearson omnibus normality test.Data sets displaying Gaussian distribution were then analyzed by One-way ANOVA with Tukey's test for multiple comparisons.Data sets displaying non-Gaussian distribution were analyzed by Kruskal-Wallis test with Dunn's test for multiple comparisons.All statistical analyses were performed using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA).

Fig 1 .
Fig 1.The type II secretion associated gene loci as found in A. nosocomialis strain M2.Genes predicted to encode proteins required for the biogenesis of a functioning type II secretion system were clustered into five distinct loci and were distantly spread over the chromosome.A single prepilin/prepseudopilin peptidase homolog was identified, which was located in the previously described pilBCD cluster[45].

Fig 2 .
Fig 2. Identification of putative type II secreted proteins from A. nosocomialis strain M2. (A) Secreted protein fractions from the parent, ΔgspD::kan mutant, and the gspD complemented strain were analyzed by one dimensional SDS-PAGE.Red arrows indicate silver reactive bands that were present in both parent and gspD complemented strain's secreted fractions.(B) Secreted protein fractions from the parent strain and the ΔgspD::kan mutant were analyzed by two-dimensional difference gel electrophoresis (2D-DIGE).A representative gel image showing Cy3 (ΔgspD::kan, green) and Cy5-labeled (parent, red) proteins that were isoelectric focused on pH strips (3-10), separated by size using SDS-PAGE, and visualized using a Typhoon 9400 variable mode imager.A merged image of the Cy3 and Cy5-labeled proteins is shown.Proteins with greater abundance in the ΔgspD::kan sample appear green and proteins with greater abundance in the parent strain sample appear red.Proteins that did not change relative abundance between the two samples appear yellow.(C) Putative T2S-dependent proteins identified via 2D-DIGE analyses.Protein candidates associated with the largest spot fold change were bioinformatically examined for the presence of a signal peptide and putative functions.1The protein score is derived from Mascot and provides an indication of how well the peptides matched the indicated protein sequence.The actual score is calculated by the following equation: protein score = -10*Log(P), where P is the probability that the protein match is a random event.Scores above 100 indicate that p < 0.05.2The protein match score indicates the number of unique peptides that matched the sequence of the identified protein.Two unique peptide matches to a protein sequence confirms the identity of a protein.

Fig 3 .
Fig 3.The predicted major pseudopilin, GspG, was processed by the prepilin peptidase PilD.Whole cell lysates from strains containing either the empty vector or pWH-gspG-FLAG were examined by western blot and probed for processed and unprocessed GspG-FLAG.GspG-FLAG from both the parent and pilD complemented strain migrated at a faster electrophoretic mobility when compared to GspG-FLAG from the ΔpilD::kan mutant.The theoretical molecular mass of full length GspG and processed GspG is 18,549 Daltons and 14,360 Daltons, respectively.doi:10.1371/journal.ppat.1005391.g003

Fig 4 .
Fig 4. Secretion of the CpaA metallopeptidase was reliant upon both a functioning T2SS and the novel CpaB chaperone.(A) Gene arrangement of the cpaAB gene cluster.(B) DELTA BLASTp analysis of the CpaB amino acid sequence identified a SRPBCC domain.(C) Western blot analysis on whole cell lysates and secreted protein fractions probing for CpaA-His.All strains and fractions were also analyzed for RNA polymerase expression, which served as a lysis control.CpaA-His expression was detected in all strains carrying pWH-cpaA-his; however, CpaA-His secretion was only detected in strains predicted to produce a functioning T2SS.(D) Western blot analysis on whole cell lysates and secreted protein fractions probing for CpaA-His from the parent strain, the ΔcpaB::frt mutant, and the cpaB complemented strain.CpaA-His was detected in all strains; however, CpaA-His secretion was not detected in the

Fig 5 .
Fig 5.Secretion of a LipA lipase ortholog was dependent on the type II secretion system and the LipB chaperone.(A) Gene arrangement of the lipBA gene cluster.(B) BLASTp analysis of the LipB amino acid sequence identified multiple domains associated with steric chaperone activity including the LimK domain and a lipase chaperone superfamiliy domain.(C) Western blot analysis on whole cell lysates and secreted protein fractions probing for LipA-His.All strains and fractions were also analyzed for RNA polymerase expression, which served as a lysis control.LipA-His expression was detected in all strains carrying pWH-lipA-his as well as pWH-lipB-lipA-his; however, LipA-His secretion was only detected in strains co-expressing the chaperone LipB as well as a predicted functioning T2SS.(D) Secretion of LipA-His was independent of the type IV pilus, as indicated by active secretion in the ΔpilA::frt mutant.(E) Western blot analysis on whole cell lysates or secreted protein fractions from A. baumannii ATCC 19606 carrying the empty vector, the pWH-lipA-his vector, or the pWH-lipB-lipA-his vector.LipA-His expression was detected; however, LipA-His secretion was only detected when the lipB gene was co-expressed with lipA-His.(F) Lipolytic activity of purified culture supernatants from the indicated strains as determined by a modified p-NPP assay.Increases in the A 410 indicate lipolytic activity.A 410 measurements were recorded every 30 minutes for 12 hours.Three biological replicates with three technical replicates were used for analysis.
Fig 7.The T2SS of A. nosocomialis strain M2 is required for optimal virulence in the G. mellonella infection model.(A) Groups of G. mellonella larvae were injected with 10μL of either the parent strain, the ΔgspD::kan mutant, or the complemented gspD::kan strain at an inoculum previously determined to be the equivalent of the LD 50 for the parent strain.Larvae were checked for viability as determined by melanin accumulation and motility.(B) Groups of G. mellonella were injected with 10μL of either the parent strain, the ΔgspD::kan mutant, or the complemented gspD::kan strain at an inoculum previously determined to be the LD 50 for the ΔgspD::kan mutant.Larvae were checked for viability as determined by melanin accumulation and motility.Survival curves were determined to be statistically significant using the Mantel-Cox test (P = 0.0147).

Fig 8 .
Fig 8.A. nosocomialis strain M2 required its T2SS for optimal colonization of both the lungs and spleen.Mice were intranasally inoculated with 1X10 9 CFU of either the wild type, ΔgspD::frt mutant, or the gspD complemented strain.After 36h post-infection, mice were sacrificed, organs harvested, and CFUs enumerated from homogenized tissue.(A) Total CFUs from the lung demonstrated a statistically significant difference between the complemented gspD strain and the ΔgspD::frt mutant (Kruskal-Wallis non-parametric with Dunn's multiple comparison test, ANOVA, *p = 0.0467).(B) Total CFUs from the spleen demonstrated statistically significance comparisons for both comparisons of either the wild type and the ΔgspD::frt mutant as well as the complemented gspD strain and the ΔgspD::frt mutant (One way ANOVA, **p = 0.0230, ***p = 0.0078).Open boxes indicate CFUs that were below the limit of detection.(C) Total CFUs form the liver did not demonstrate any statistically significant differences.Open boxes indicate CFUs that were below the limit of detection