Stringent Response Governs the Virulence and Oxidative Stress Resistance of Francisella tularensis

Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its ability to cause severe illness upon inhalation of as few as ten organisms and based on its potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by the CDC. The stringent response facilitates bacterial survival under nutritionally challenging starvation conditions. The hallmark of stringent response is the accumulation of the effector molecules ppGpp and (p)ppGpp known as stress alarmones. The relA and spoT gene products generate alarmones in several Gram-negative bacterial pathogens. RelA is a ribosome-associated ppGpp synthetase that gets activated under amino acid starvation conditions whereas, SpoT is a bifunctional enzyme with both ppGpp synthetase and ppGpp hydrolase activities. Francisella encodes a monofunctional RelA and a bifunctional SpoT enzyme. Previous studies have demonstrated that stringent response under nutritional stresses increases expression of virulence-associated genes encoded on Francisella Pathogenicity Island. This study investigated how stringent response governs the oxidative stress response of F. tularensis. We demonstrate that RelA/SpoT-mediated ppGpp production alters global gene transcriptional profile of F. tularensis in the presence of oxidative stress. The lack of stringent response in relA/spoT gene deletion mutants of F. tularensis makes bacteria more susceptible to oxidants, attenuates survival in macrophages, and virulence in mice. Mechanistically, we provide evidence that the stringent response in Francisella contributes to oxidative stress resistance by enhancing the production of antioxidant enzymes. Importance The unique intracellular life cycle of Francisella in addition to nutritional stress also exposes the bacteria to oxidative stress conditions upon its brief residence in the phagosomes, and escape into the cytosol where replication takes place. However, the contribution of the stringent response in gene regulation and management of the oxidative stress response when Francisella is experiencing oxidative stress conditions is not known. Our results provide a link between the stringent and oxidative stress responses. This study further improves our understanding of the intracellular survival mechanisms of F. tularensis.


Abstract 27
Francisella tularensis is a Gram-negative bacterium responsible for causing tularemia in 28 the northern hemisphere. F. tularensis has long been developed as a biological weapon due to its 29 ability to cause severe illness upon inhalation of as few as ten organisms and based on its 30 potential to be used as a bioterror agent is now classified as a Tier 1 Category A select agent by  relAspoT mutants to produce ppGpp, the bacterial strains were grown to late exponential 139 phase, and ppGpp production was determined by HPLC. The production of ppGpp was dropped 140 to 45 and 28% in the relA and the relAspoT mutants respectively, as compared to that 141 observed for the wild type F. tularensis LVS, indicating that relA and more so the relAspoT 142 mutant is deficient in ppGpp production ( Fig. 1C and D). The growth characteristics of the wild 143 type F. tularensis LVS, the relA, the relAspoT double mutant and the transcomplemented 144 strains were determined by growing in MH-broth in the absence or presence of 500µM of serine 145 hydroxamate, a serine homolog responsible for inducing amino acid starvation-like conditions.

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The relA mutant did not exhibit any growth defect and grew similarly to the wild type F. 147 tularensis LVS when grown in the MH-broth. The relAspoT mutant showed retarded growth 148 and entered the stationary phase after 16 hours of growth. Transcomplementation with spoT gene 149 restored the growth of the relAspoT mutant as well as prevented its early entry into the 150 stationary phase (Fig 1E). Growth of all Francisella strains was slightly reduced in the presence 151 of serine hydroxamate; however, the relAspoT mutant failed to grow in its presence.

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Transcomplementation of the relAspoT with the spoT gene restored its growth in the presence 153 of serine hydroxamate (Fig. 1F). Sensitivities of the wild type F. tularensis LVS, the relA, the 154 relAspoT mutants and the transcomplemented strains were also tested against streptomycin, 155 nitrofurantoin, and tetracycline by disc diffusion assays. It was observed that the relAspoT 156 mutant exhibited enhanced sensitivities towards all the three antibiotics tested as compared to the 157 wild type F. tularensis LVS or the relA mutant. Transcomplementation of the relAspoT 158 mutant restored the sensitivities similar to those observed for the wild type or the relA mutant 159 (Fig. 1G). Collectively, these results demonstrate that induction of RelA/SpoT-mediated 160 stringent response associated with the production of ppGpp is required for growth of F.  (Fig. 2). Treatment of the relAspoT mutant with H 2 O 2 resulted in differential 177 expression of a total of 855 genes in the relAspoT mutant as compared to the wild type F. 178 tularensis LVS. Of the 450 (53%) downregulated genes, majority of the genes belonged to 179 metabolism (n=155), hypothetical proteins (n=95), others (n=49) and FPI (n=32) categories. The 180 genes that were upregulated following exposure to H 2 O 2 also belonged to the similar categories 181 except for the FPI genes (Fig. 2). Collectively these results demonstrate that RelA and SpoT not 182 only control the expression of several genes of F. tularensis under normal growth conditions but 183 also regulate genes both positively and negatively when the bacteria are exposed to the oxidative 184 stress. 186 We further analyzed the differential expression of the FPI genes in the RelA/SpoT positively regulates the expression of virulence-associated genes encoded on the FPI 199 under normal as well as oxidative stress conditions (Fig. 3A). 200 We also confirmed the expression profiles of select FPI genes in the wild type F.  relAspoT mutants restored the intramacrophage replication (Fig. 3D). 219 We also investigated if the loss of relA and relA spoT is associated with the attenuation of 220 virulence in mice. It was observed that 100% of C57BL/6 mice infected intranasally either with 221 1×10 5 or 1×10 6 CFUs of the relA or the relAspoT mutant survived the infection. Mice these genes were found to be downregulated in the relAspoT mutant as compared to the wild 237 type F. tularensis LVS when exposed to the oxidative stress. All these three genes are 238 transcribed as single transcription units. Genes encoding Clp ATPases clpP, clpX, and lon; 239 hslU, hslV; grpE, dnaK, and dnaJ were downregulated in untreated relAspoT mutant and were 240 further downregulated when exposed to the oxidative stress induced by H 2 O 2. A group of genes 241 encoding the universal stress protein, protease sohB, and starvation protein A were down-242 regulated in untreated relAspoT mutant and remained downregulated upon exposure to the 243 oxidative stress (Fig. 4A). The differential expression of these genes was also confirmed by qRT-244 PCR. The expression profile of the representative stress response genes was similar to that 245 observed by RNAseq. However, all these genes were significantly downregulated in untreated as 246 well as H 2 O 2 treated relAspoT mutant as compared to the F. tularensis LVS.

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Transcomplementation of the relA mutant restored the wild type phenotype, while only a partial 248 restoration of the gene expression was observed in the relAspoT mutant (Fig. 4B).

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Since RelA-SpoT-dependent induction of heat shock proteins is associated with survival 250 at higher temperatures, we also examined the effect of the loss of relA and relA/spoT on bacterial 251 viability when exposed to a higher temperature of 48  C. It was observed that the viability of the 252 relA mutant was unaffected and remained similar to the wild type F. tularensis when exposed 253 to a temperature of 48  C for 1 hour and 5-7 fold fewer bacteria were recovered after 3 hours of 254 exposure. On the other hand, nearly 10-20-fold less viable bacteria were recovered when the 255 relAspoT mutant was exposed to a temperature of 48  C for 1 and 3 hours, respectively.

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Transcomplementation of the relAspoT mutant restored the wild type phenotype (Fig. 4D).   . We further investigated if the wild type phenotype is restored in the 360 relAspoT+pkatG and the relAspoT+psodB strains when exposed to superoxide-generating 361 compounds and organic peroxides. It was observed that exposure of the relAspoT+pkatG 362 strain to paraquat and TBH restored the wild type phenotype; while only a partial restoration was 363 observed against menadione (Fig. 9A, B and C). On the other hand the wild type phenotype of 364 the relAspoT+psodB strain was restored only to menadione, but not when exposed either to 365 paraquat or TBH (Fig. 9D, E and F). These results indicate that both KatG and SodB may be 366 required for full restoration of the wild type phenotype in overcoming the oxidative stresses. It 367 has previously been reported that stringent response mediates antibiotic resistance by modulating  Unlike previous studies that mostly used amino acid starvation as a means to induce 429 stringent response, in this study, we investigated the role of stringent response during the 430 exponential stage of bacterial growth in a nutritionally rich environment and when the bacteria 431 are exposed to oxidative stress. We observed that ppGpp production was reduced drastically in 432 both the relA and the relAspoT mutants as compared to wild type F. tularensis LVS even 433 when the bacteria were still in the exponential phase of growth and not exposed to any stress, 434 indicating that expression of ppGpp is also required under homeostatic growth conditions. Our   Table 1.  Table 1.

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The ppGpp extraction was performed as previously described (23). Briefly, the bacterial     Table 2.      Figure 6 A B