Differential Growth of Francisella tularensis, Which Alters Expression of Virulence Factors, Dominant Antigens, and Surface-Carbohydrate Synthases, Governs the Apparent Virulence of Ft SchuS4 to Immunized Animals

The gram-negative bacterium Francisella tularensis (Ft) is both a potential biological weapon and a naturally occurring microbe that survives in arthropods, fresh water amoeba, and mammals with distinct phenotypes in various environments. Previously, we used a number of measurements to characterize Ft grown in Brain-Heart Infusion (BHI) broth as (1) more similar to infection-derived bacteria, and (2) slightly more virulent in naïve animals, compared to Ft grown in Mueller Hinton Broth (MHB). In these studies we observed that the free amino acids in MHB repress expression of select Ft virulence factors by an unknown mechanism. Here, we tested the hypotheses that Ft grown in BHI (BHI-Ft) accurately displays a full protein composition more similar to that reported for infection-derived Ft and that this similarity would make BHI-Ft more susceptible to pre-existing, vaccine-induced immunity than MHB-Ft. We performed comprehensive proteomic analysis of Ft grown in MHB, BHI, and BHI supplemented with casamino acids (BCA) and compared our findings to published “omics” data derived from Ft grown in vivo. Based on the abundance of ~1,000 proteins, the fingerprint of BHI-Ft is one of nutrient-deprived bacteria that—through induction of a stringent-starvation-like response—have induced the FevR regulon for expression of the bacterium's virulence factors, immuno-dominant antigens, and surface-carbohydrate synthases. To test the notion that increased abundance of dominant antigens expressed by BHI-Ft would render these bacteria more susceptible to pre-existing, vaccine-induced immunity, we employed a battery of LVS-vaccination and S4-challenge protocols using MHB- and BHI-grown Ft S4. Contrary to our hypothesis, these experiments reveal that LVS-immunization provides a barrier to infection that is significantly more effective against an MHB-S4 challenge than a BHI-S4 challenge. The differences in apparent virulence to immunized mice are profoundly greater than those observed with primary infection of naïve mice. Our findings suggest that tularemia vaccination studies should be critically evaluated in regard to the growth conditions of the challenge agent

Host-Adaptation of Francisella tularensis Alters the Bacterium's Surface-Carbohydrates to Hinder Effectors of Innate and Adaptive Immunity

The gram-negative bacterium Francisella tularensis survives in arthropods, fresh water amoeba, and mammals with both intracellular and extracellular phases and could reasonably be expected to express distinct phenotypes in these environments. The presence of a capsule on this bacterium has been controversial with some groups finding such a structure while other groups report that no capsule could be identified. Previously we reported in vitro culture conditions for this bacterium which, in contrast to typical methods, yielded a bacterial phenotype that mimics that of the bacterium's mammalian, extracellular phase.SDS-PAGE and carbohydrate analysis of differentially-cultivated F. tularensis LVS revealed that bacteria displaying the host-adapted phenotype produce both longer polymers of LPS O-antigen (OAg) and additional HMW carbohydrates/glycoproteins that are reduced/absent in non-host-adapted bacteria. Analysis of wildtype and OAg-mutant bacteria indicated that the induced changes in surface carbohydrates involved both OAg and non-OAg species. To assess the impact of these HMW carbohydrates on the access of outer membrane constituents to antibody we used differentially-cultivated bacteria in vitro to immunoprecipitate antibodies directed against outer membrane moieties. We observed that the surface-carbohydrates induced during host-adaptation shield many outer membrane antigens from binding by antibody. Similar assays with normal mouse serum indicate that the induced HMW carbohydrates also impede complement deposition. Using an in vitro macrophage infection assay, we find that the bacterial HMW carbohydrate impedes TLR2-dependent, pro-inflammatory cytokine production by macrophages. Lastly we show that upon host-adaptation, the human-virulent strain, F. tularensis SchuS4 also induces capsule production with the effect of reducing macrophage-activation and accelerating tularemia pathogenesis in mice.F. tularensis undergoes host-adaptation which includes production of multiple capsular materials. These capsules impede recognition of bacterial outer membrane constituents by antibody, complement, and Toll-Like Receptor 2. These changes in the host-pathogen interface have profound implications for pathogenesis and vaccine development

The Many Roles of the Bacterial Second Messenger Cyclic di-AMP in Adapting to Stress Cues

Bacteria respond to changes in environmental conditions through adaptation to external cues. Frequently, bacteria employ nucleotide signaling molecules to mediate a specific, rapid response. Cyclic di-AMP (c-di-AMP) was recently discovered to be a bacterial second messenger that is essential for viability in many species. In this review, we highlight recent work that has described the roles of c-di-AMP in bacterial responses to various stress conditions. These studies show that depending on the lifestyle and environmental niche of the bacterial species, the c-di-AMP signaling network results in diverse outcomes, such as regulating osmolyte transport, controlling plant attachment, or providing a checkpoint for spore formation.</jats:p

Systematic identification of molecular mediators underlying sensing of<i>Staphylococcus aureus</i>by<i>Pseudomonas aeruginosa</i>

ABSTRACTBacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogensPseudomonas aeruginosaandStaphylococcus aureusthat are frequently found in co-infections. We discovered thatP. aeruginosasenses diverse staphylococcal exoproducts including the metallophore staphylopine, intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. Further, we show that staphylopine inhibits biofilm formation and thatP. aeruginosacan utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Our screening approach thus identified multipleS. aureussecreted molecules that are sensed byP. aeruginosaand affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species.</jats:p

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens

Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here, we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in coinfections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine (StP), intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. We observed that StP inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Due to the unbiased nature of our approach, we also identified on a genome scale the genes in S. aureus that affect production of each sensed exoproduct, providing possible targets to modify multispecies community dynamics. Further, a combination of these identified S. aureus products recapitulated a majority of the transcriptional response of P. aeruginosa to S. aureus supernatant, validating our screening strategy. Cystic fibrosis (CF) clinical isolates of both S. aureus and P. aeruginosa also showed varying degrees of induction or responses, respectively, which suggests that these interactions are widespread among pathogenic strains. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species.</jats:p

Systematic identification of molecular mediators of interspecies sensing in a community of two frequently coinfecting bacterial pathogens.

Bacteria typically exist in dynamic, multispecies communities where polymicrobial interactions influence fitness. Elucidating the molecular mechanisms underlying these interactions is critical for understanding and modulating bacterial behavior in natural environments. While bacterial responses to foreign species are frequently characterized at the molecular and phenotypic level, the exogenous molecules that elicit these responses are understudied. Here, we outline a systematic strategy based on transcriptomics combined with genetic and biochemical screens of promoter-reporters to identify the molecules from one species that are sensed by another. We utilized this method to study interactions between the pathogens Pseudomonas aeruginosa and Staphylococcus aureus that are frequently found in coinfections. We discovered that P. aeruginosa senses diverse staphylococcal exoproducts including the metallophore staphylopine (StP), intermediate metabolites citrate and acetoin, and multiple molecules that modulate its iron starvation response. We observed that StP inhibits biofilm formation and that P. aeruginosa can utilize citrate and acetoin for growth, revealing that these interactions have both antagonistic and beneficial effects. Due to the unbiased nature of our approach, we also identified on a genome scale the genes in S. aureus that affect production of each sensed exoproduct, providing possible targets to modify multispecies community dynamics. Further, a combination of these identified S. aureus products recapitulated a majority of the transcriptional response of P. aeruginosa to S. aureus supernatant, validating our screening strategy. Cystic fibrosis (CF) clinical isolates of both S. aureus and P. aeruginosa also showed varying degrees of induction or responses, respectively, which suggests that these interactions are widespread among pathogenic strains. Our screening approach thus identified multiple S. aureus secreted molecules that are sensed by P. aeruginosa and affect its physiology, demonstrating the efficacy of this approach, and yielding new insight into the molecular basis of interactions between these two species

Intermediate metabolites produced by <i>S</i>. <i>aureus</i> are sensed and utilized as carbon sources by <i>P</i>. <i>aeruginosa</i>.

(A) Transposon library screen with P′acoR-reporter strain to determine mutations in S. aureus that increase or decrease induction in P. aeruginosa. For each mutant supernatant, the z-scores calculated from the mean slope (RFU/OD600/hr) of each respective experiment for 2 replicates of the screen are shown (z-score 1, up-regulating, blue). (B) RFU per OD600 of P′acoR-mScarlet after addition of media, S. aureus supernatant, or the indicated concentrations of acetoin. (C) Enzymatic reactions of butanoate metabolism are shown, with the colors representing the effect of the mutant supernatants on promoter induction (down-regulating, red; up-regulating, blue; mutant not in library and/or no change, black). (D) Acetoin measurements of the up-regulating and down-regulating supernatants correlate with P′acoR induction (r = 0.2157). Acetoin concentration data were analyzed by 1-way ANOVA with Dunnett test for multiple comparisons to the WT control. For panels B and D, data shown represent 3 independent replicates and error bars denote the SD. (E) Growth of P. aeruginosa was monitored at OD600 after the addition of 1 mM glucose, citric acid, 2,3-butanediol, acetoin, or a mix of citric acid, 2,3-butanediol, and acetoin as the sole carbon sources. Two independent replicates are shown. Shaded regions denote the SD. The data underlying panels AC and D can be found in Tables K and L in S1 File, and data for panels B and E can be found in S1 Table. RFU, relative fluorescence unit; SD, standard deviation; WT, wild type.</p

Excel file containing numerical data underlying Figs 2B, 3B, 3C,3D, 3E, 3F, 3G, 4C, 4D, 4E, 5D, 6B, 6E, 7A, 7B, 7C, 7D, 8C, S2A, S2B, S2C, S3A, S3B, S3C, S3D, S4A, S4B, S4C, S5A, S5B, S6, S7 and S8B.

(XLSX)</p

<i>P</i>. <i>aeruginosa</i> promoter-reporters are induced by <i>S</i>. <i>aureus</i> exoproducts.

(A) Expression levels from P. aeruginosa RNA-seq after exposure to S. aureus supernatant or media control for four transcripts as gene candidates for promoter-reporter construction. Gene annotations: PA14_11320, Zur-regulon putative lipoprotein in operon with ABC transporter; pvdG, pyoverdine biosynthesis thioesterase; opdH, TCA uptake porin; acoR, transcriptional regulator of acetoin catabolism genes. (B) RFUs of mScarlet normalized to OD600 over time after exposure to S. aureus supernatant or media control in the indicated promoter-reporter strains. Slope calculated from 1.5 to 5 hours (promoters of PA14_11320, opdH, and acoR) or 1 to 4 hours (promoter of pvdG) after addition of supernatant, supernatant dilutions, or media control. Each strain had higher expression of mScarlet after supernatant addition compared with media control and displayed dose-dependent responses. Shaded regions and error bars show the SD. The data underlying panels A and B can be found in Table A in S1 File and S1 Table, respectively. RFU, relative fluorescence unit; RNA-seq, RNA sequencing; SD, standard deviation.</p

StP and zinc levels affect <i>S</i>. <i>aureus</i> and <i>P</i>. <i>aeruginosa</i> physiology.

(A) WT S. aureus and NTML transposon mutants were grown in media with or without the addition of the zinc chelator TPEN at the concentrations indicated. Growth was measured at OD600 after 24 hours. Datasets were analyzed by a 2-way ANOVA with Dunnett test for multiple comparisons to the respective WT value. (B, C) RFU of mScarlet normalized to OD600 over time after exposure to media control or S. aureus supernatant and/or addition of the indicated concentrations of (B) TPEN or (C) zinc, nickel, and cobalt in the indicated P. aeruginosa promoter-reporter strains (promoters of PA14_11320, cntO, or dksA2). Datasets were analyzed by a 2-way ANOVA with Tukey test for multiple comparisons. Statistics shown represent tests comparing (B) the respective conditions/promoters with and without TPEN and (C) all treatments to the respective supernatant addition. (D) S. aureus cultures were grown in culture with 50% medium salts (Control) or cell-free supernatants of P. aeruginosa grown in the presence of the indicated additives. Colony-forming units per milliliter of S. aureus were calculated after 16 hours of growth. Datasets were analyzed by 1-way ANOVA with Dunnett test for multiple comparisons to the S. aureus grown in supernatant from P. aeruginosa grown in media without additions. Data shown for all panels are the means of 3 independent biological replicates, including 3 separate supernatant collections in D. The error bars denote the SD. *, p p p p S1 Table. NTML, Nebraska Transposon Mutant Library; RFU, relative fluorescence unit; SD, standard deviation; StP, staphylopine; WT, wild type. (TIF)</p