Table 1.
Mutants in mini-TnSeq assay.
Fig 1.
Virulence of mutants in mini-TnSeq assay.
Fitness of knockouts generated in YPIII/pIB1- (A, C) or IP2666/pIB1+ (B, D) at 3 days post-intravenous infection with 104 and 103, respectively, of mini-TnSeq libraries. Fitness values were obtained by dividing the proportion of sequencing reads for a mutant in the liver (A, B) or spleen (C, D) by its proportion of reads in the inoculum. Each data point for a mutant represents an individual mouse. N = 7–10 mice. Fitness values were log10 transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test. P-values represent comparisons between the fitness scores of individual mutants with the fitness score of the two knockouts of neutral loci (pooled together) in each respective condition. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and **** indicates p≤0.0001.
Fig 2.
Mini-TnSeq accurately predicted outcomes of 1:1 competition experiments.
Mice were inoculated intravenously with 1:1 mixture of 103 bacteria, comprised of WT-KanR and a drug-sensitive mutant. Livers (A) and spleens (B) were dissected at 3 days post-infection, homogenized and plated onto selective and non- selective plates to determine the competitive index (C.I.). Each symbol represents an individual mouse. N = 4–10. C.I. data was log10 transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test comparing the C.I. values of mutant strains with those of the WT strain. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and **** indicates p≤0.0001.
Fig 3.
Four mutants displayed fitness changes following phagocytic cell depletion.
Mice were intraperitoneally injected with RB6-8C5 (A-B) or 1A8 (C-D) 24 hours prior to and post-infection. Mice were inoculated intravenously with 103 CFU of the IP2666/pIB1+ mutant library. Fitness values were obtained by dividing the proportion of sequencing reads for a mutant in the depleted liver (A, C) or spleen (B, D) by its proportion of reads in the inoculum. Each data point for a mutant represents an individual mouse. N = 5–10 mice. Non-depleted fitness values are the same is in Fig 1. Fitness scores values were log10 transformed and an unpaired t-test with the Holm-Sidak correction for multiple comparisons was performed to calculate statistically significant differences between the fitness scores of specific bacterial mutants in depleted versus non-depleted mice. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and **** indicates p≤0.0001.
Fig 4.
ΔdusB-fis is sensitive to neutrophils and inflammatory monocytes in vivo.
C57BL6/J mice were inoculated intravenously with a pool of 103 bacteria, containing an equal mixture of WT and WT-KanR, Δfis-KanR and WT, ΔdusB-fis and WT-KanR, or ΔdusB-fis::dusB-fis and WT-KanR. 24 hours prior to and post-infection, some mice were intraperitoneally injected with either the 1A8 or RB6-8C5 antibody. Other mice were intraperitoneally injected with the MC-21 antibody 1 day prior to infection and each day after until completion of the experiment. Mice were euthanized at the indicated times and livers (A) and spleens (B) were collected, and dilutions of tissue homogenates were plated onto selective and non-selective media to determine the C.I. C.I. data was log10 transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test comparing the C.I. values of mutant strains to WT or C.I. values in non-depleted and depleted mice. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and **** indicates p≤0.0001.
Fig 5.
ΔdusB-fis can colonize, but is unable to sustain growth in systemic tissue sites.
C57BL6/J mice were inoculated intravenously with a pool of 103 bacteria, containing either an equal mixture of WT yopE::mcherry and ΔdusB-fis-KanR (A-B), or with WT yopE::mcherry only or ΔdusB-fis-KanR only (C-D). At 4-hour, 24-hour, 48-hour, and 72-hour time-points post-infection, mice were euthanized and spleens and livers were collected. Dilutions of liver (A, C) and spleen (B, D) homogenates were plated and CFU calculated. In co-infection experiments (A-B), the number of bacteria recovered from selective and non-selective plates was used to determine quantity of WT yopE::mcherry and ΔdusB-fis-KanR bacteria in each organ sample. CFU/g data was log10 transformed and the values of ΔdusB-fis-KanR to WT yopE::mcherry bacteria in each tissue at each time point was compared using an unpaired t-test with the Holm-Sidak correction for multiple comparisons. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001, and **** indicates p≤0.0001.
Fig 6.
ΔdusB-fis is not defective for Type 3 Secretion or effector translocation.
A) Stationary phase cultures of WT, ΔyscF, ΔdusB-fis, and Δfis strains were diluted 1:40 into L broth with 20mM sodium oxalate and 20 mM MgCl2. Cultures were grown for 2 hours at 26°C and then shifted to 37°C for 2 hours. After growth, proteins from culture supernatants were precipitated, resolved on a polyacrylamide gel alongside a standard protein ladder, and visualized using commaasie blue. B-C) Stationary phase cultures of WT, ΔyscF, and ΔdusB-fis strains containing YopH-TEM (HTEM) or YopE-TEM (ETEM) beta-lactamase reporters were grown as in A. After growth, the culture supernatant was mixed with nitrocefin at a final concentration of 100 μg/mL. After 10 minutes of incubation, the A490 of samples was measured using a BioTek Synergy HT plate reader. Statistical significance was calculated using One Way ANOVA analysis with the Dunnett’s multiple comparison test on log10-transformed values. Each bar represents the mean and standard error of 3 biological replicates. D-E) Stationary phase cultures of WT, ΔyopB, and ΔdusB-fis strains fused containing H-TEM or E-TEM beta-lactamase reporters were grown as in A-C. HEp-2 cells were infected at a multiplicity of infection of 10 for 1 hr and then incubated with CCF4 to determine the percentage of cells containing translocated effectors (% blue). Data was log10 transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test comparing each strain to WT. Each bar represents the mean and standard error of 3–6 biological replicates. F-G) C57BL6/J mice were inoculated intravenously with a pool of 103 bacteria, containing an equal mixture of ΔdusB-fisΔyscF -KanR and ΔyscF. 24 hours prior to and post-infection, mice were intraperitoneally injected with RB6-8C5 antibody. Mice were euthanized at 3 days post-infection and livers (F) and spleens (G) were collected, and dilutions of tissue homogenates were plated onto selective and non-selective media to determine the C.I. C.I. values were log10 transformed and statistical significance was calculated using a Mann-Whitney t-test. * indicates p≤0.05, ** indicates p≤0.01, **** indicates p≤0.0001.
Fig 7.
dusB-fis is required for resistance to oxidative stress.
A) Stationary phase cultures of WT and ΔdusB-fis were diluted 1:100 into L broth and L broth adjusted to pH 5.5 and the OD600 of cultures was measured at 1-hour-intervals for 12 hours during growth with aeration. Each symbol represents the mean of 2–4 biological replicates. B) Stationary phase cultures were diluted 1:100 into a well of a 96-well plate containing L broth or L broth + 250 μM 2,2’- Bipyridyl and OD600 measurements were recorded at 15-minute intervals during growth with aeration. Lines represent the mean of 3 biological replicates. (C-D) Exponential phase cultures were washed and diluted 1:50 into M9 glucose medium or M9 glucose containing 2.5mM DETA NONOate (C) or M9 glucose containing 1.5mM H2O2 (D) for 60 minutes. Survival was calculated by determining the number of CFUs recovered following treatment divided by the number of CFUs recovered from untreated cultures. The mean and standard error of 3 biological replicates for DETA NONOate treatment and 6–10 biological replicates for H2O2 treatment are shown. Survival values were log10 transformed and statistical significance was calculated using One Way ANOVA analysis with Dunnett’s multiple comparison post-test comparing each strain to WT. E) Δfis fails to up-regulate ROS-responsive genes after exposure to H2O2. Exponential phase cultures were washed and diluted 1:50 into M9 glucose medium or M9 glucose containing 20 μM H2O2 and were incubated for 10 minutes with aeration. RNA isolated from treated and untreated samples was used to generate cDNA, and qPCR reactions were performed. Relative expression was determined by normalizing to 16S RNA as well as to expression in untreated samples using the ΔΔCT method. Bars represent the mean and standard error of 8 biological replicates. Unpaired Mann-Whitney t-tests were performed to calculate statistical differences between expression of each gene in WT and Δfis. * indicates p≤0.05, ** indicates p≤0.01, **** indicates p≤0.0001, ns indicates not significant.
Fig 8.
ΔdusB-fis is sensitive to ROS produced by the NADPH oxidase complex during mouse infection.
C57Bl/6 or C57Bl/6 gp91phox-/- mice were inoculated intravenously with 1x103 CFU of a 1:1 mixture of WT and ΔdusB-fis-KanR. Livers (A,C,E) and spleens (B,D,F) were collected, weighed, homogenized, and plated for CFUs on selective and non-selective agar at 3 days-post-infection. (A-B) The number of bacteria recovered from selective and non-selective plates was used to determine the C.I. of ΔdusB-fis-KanR. Each data point represents an individual mouse. C.I. data was log10 transformed and statistical significance was calculated using the Mann-Whitney t-test. (C-D) The CFU/gram was determined by dividing the total number of recovered CFU on non-selective plates by the weight of the tissue. CFU/g data was log10 transformed and statistical significance was calculated using the Mann-Whitney t-test. (E-F) The number of bacteria recovered from selective and non-selective plates was used to determine the quantity of ΔdusB-fis-KanR and WT bacteria in each organ sample. CFU/g data was log10 transformed and statistical significance was calculated using One Way ANOVA analysis with the Dunnett’s multiple comparison test. * indicates p≤0.05, ** indicates p≤0.01, *** indicates 00.001, **** indicates p≤0.0001.