Adaptive Mechanisms of Niche Remodeling in Streptococcus pyogenes

The Gram-positive bacterium Streptococcus pyogenes is a remarkably successful pathogen, capable of infecting numerous tissue sites within its human host. The ability of S. pyogenes to invade these different niches is, in part, due to the species’ ability to monitor various physical and chemical signals in its environment and alter its transcriptional profile in response to these differential conditions. As a member of the lactic acid bacteria, S. pyogenes has a simple fermentative metabolism and relies exclusively on a combination of homo-lactic and mixed acid fermentation as a means of generating energy in the cell. As a consequence of its fermentative metabolism, S. pyogenes produces several organic acid end products that, over time, accumulate in the surrounding environment, causing a substantial reduction in pH. Thus, growth of the bacterium itself results in a significant remodeling of its local tissue environment. It also indicates that over the course of infection, it must both adapt to its self-inflicted acid stress as well as exploit alternative carbon sources for survival. Although pH has been identified as a signal utilized by S. pyogenes to induce global transcriptional changes, the specific regulatory mechanisms behind this transcriptional remodeling have largely remained unclear. To further characterize the process of S. pyogenes’ pH adaptive response we have identified several novel pH-sensitive transcriptional regulators and analyzed their contribution to gene expression and S. pyogenes pathogenesis. The malic enzyme pathway, which allows the cell to utilize malate as a carbon source for growth, consists of four genes in two adjacent operons, with the regulatory TCS MaeKR being required for the expression of the genes encoding a malate permease (maeP) and malic enzyme (maeE). Results show that expression of the maePE operon is influenced independently by external malate concentrations and pH in a MaeK-dependent mechanism. The ME genes are additionally regulated by a unique CcpA-independent form of catabolite repression which involves the PTS proteins PtsI and HPr. Furthermore, in vivo experiments demonstrate that loss of any individual ME gene has a significant effect on the outcome of a soft tissue infection. The secreted toxins SPN and SLO have been shown to contribute to S. pyogenes cytotoxicity and virulence in multiple models of pathogenesis, however little information is known about the specific regulatory mechanism that control expression of these toxins. Our work has determined that the growth-phase pattern of expression of the spn/slo operon is regulated by environmental pH. Additionally, this regulation requires both the CovRS two-component system as well as an additional protein, RocA. Additional data suggests that RocA does not function as a traditional histidine kinase, despite high structural and sequence homology to known histidine kinases such as CovS. However, all three regulatory proteins are required for the pH-mediated regulation of this virulence operon

Streptococcus pyogenes malate degradation pathway links pH regulation and virulence

The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is in-duced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Further-more, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI – mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP–, MaeK–, and MaeR – mu-tants were attenuated for virulence, whereas a MaeE – mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes ’ carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection. Although it has a relatively small genome (approximately 1.8Mbp), the pathogenic Gram-positive bacterium Streptococcus pyogenes has a remarkable ability to adapt to a variety of huma

Complete genome sequence of emm type 14 Streptococcus pyogenes strain HSC5

Streptococcus pyogenes causes a greater diversity of human disease than any other bacterial pathogen. Here, we present the complete genome sequence of the emm type 14 S. pyogenes strain HSC5. This strain is a robust producer of the cysteine protease SpeB and is capable of producing infection in several different animal models

Complete genome sequences of emm6 Streptococcus pyogenes JRS4 and parental strain D471

We report the complete genome assemblies of the group A Streptococcus pyogenes serotype emm6 strain D471 and its streptomycin-resistant derivative JRS4. Both of these well-studied laboratory strains have been extensively characterized over the past three decades and have been instrumental in the discovery of multiple aspects of streptococcal pathogenesis

CcpA Coordinates Growth/Damage Balance for Streptococcus pyogenes Pathogenesis

Abstract To achieve maximum fitness, pathogens must balance growth with tissue damage, coordinating metabolism and virulence factor expression. In the gram-positive bacterium Streptococcus pyogenes, the DNA-binding transcriptional regulator Carbon Catabolite Protein A (CcpA) is a master regulator of both carbon catabolite repression and virulence, suggesting it coordinates growth/damage balance. To examine this, two murine models were used to compare the virulence of a mutant lacking CcpA with a mutant expressing CcpA locked into its high-affinity DNA-binding conformation (CcpAT307Y). In models of acute soft tissue infection and of long-term asymptomatic mucosal colonization, both CcpA mutants displayed altered virulence, albeit with distinct growth/damage profiles. Loss of CcpA resulted in a diminished ability to grow in tissue, leading to less damage and early clearance. In contrast, constitutive DNA-binding activity uncoupled the growth/damage relationship, such that high tissue burdens and extended time of carriage were achieved, despite reduced tissue damage. These data demonstrate that growth/damage balance can be actively controlled by the pathogen and implicate CcpA as a master regulator of this relationship. This suggests a model where the topology of the S. pyogenes virulence network has evolved to couple carbon source selection with growth/damage balance, which may differentially influence pathogenesis at distinct tissues

Distribution of Tn-insertions along the <i>S</i>. <i>aureus</i> genome.

<p><b>A.</b> Insertions appear uniform all along the genome with no obvious gaps or hot spots. <b>B.</b> Distribution of insertions on each strand reveal no apparent strand bias. <b>C.</b> 2,132 Tn insertions were at identical TA dinucleotides but on opposite strands. The 1066 sites appear to be uniformly distributed across the entire genome.</p

Genetic requirements for <i>Staphylococcus aureus</i> nitric oxide resistance and virulence

<div><p><i>Staphylococcus aureus</i> exhibits many defenses against host innate immunity, including the ability to replicate in the presence of nitric oxide (NO·). <i>S</i>. <i>aureus</i> NO· resistance is a complex trait and hinges on the ability of this pathogen to metabolically adapt to the presence of NO·. Here, we employed deep sequencing of transposon junctions (Tn-Seq) in a library generated in USA300 LAC to define the complete set of genes required for <i>S</i>. <i>aureus</i> NO· resistance. We compared the list of NO·-resistance genes to the set of genes required for LAC to persist within murine skin infections (SSTIs). In total, we identified 168 genes that were essential for full NO· resistance, of which 49 were also required for <i>S</i>. <i>aureus</i> to persist within SSTIs. Many of these NO·-resistance genes were previously demonstrated to be required for growth in the presence of this immune radical. However, newly defined genes, including those encoding SodA, MntABC, RpoZ, proteins involved with Fe-S-cluster repair/homeostasis, UvrABC, thioredoxin-like proteins and the F₁F₀ ATPase, have not been previously reported to contribute to <i>S</i>. <i>aureus</i> NO· resistance. The most striking finding was that loss of any genes encoding components of the F₁F₀ ATPase resulted in mutants unable to grow in the presence of NO· or any other condition that inhibits cellular respiration. In addition, these mutants were highly attenuated in murine SSTIs. We show that in <i>S</i>. <i>aureus</i>, the F₁F₀ ATPase operates in the ATP-hydrolysis mode to extrude protons and contribute to proton-motive force. Loss of efficient proton extrusion in the Δ<i>atpG</i> mutant results in an acidified cytosol. While this acidity is tolerated by respiring cells, enzymes required for fermentation cannot operate efficiently at pH ≤ 7.0 and the Δ<i>atpG</i> mutant cannot thrive. Thus, <i>S</i>. <i>aureus</i> NO· resistance requires a mildly alkaline cytosol, a condition that cannot be achieved without an active F₁F₀ ATPase enzyme complex.</p></div

Δ<i>atpG</i> exhibits both elevated ATP levels and membrane potential both before and after NO· exposure.

<p><i>S</i>. <i>aureus</i> WT and Δ<i>atpG</i> were grown in TSB 5g/L glucose and were exposed to NO· mix (10mM NOC12, 1mM DEANO) at OD₆₅₀ 0.25. <b>A.</b> Prior to NO· addition and at 1-hr post addition ATP levels were determined. <b>B.</b> Prior to NO· addition and 1-hr post addition, membrane potential was quantified. (n = 2 for ATP levels, n = 3 for membrane potential). Significance was determined with two-sided Student’s t tests (*, P ≤ .05; **, P ≤ .01).</p

Δ<i>atpG</i> is attenuated during non-respiratory growth and is also sensitive to peroxide and kanamycin.

<p><b>A.</b> and <b>B.</b> <i>S</i>. <i>aureus</i> LAC WT and Δ<i>atpG</i> were grown in TSB 5g/L glucose aerobically, with 10mM DETA/NO, with 2mM 2,2’-dipyridyl, anaerobically, or anaerobically with nitrate. <b>A.</b> Maximum absorbance (650nm) reached over a 24-hr growth curve and <b>B.</b> maximum growth rate reached over a 24-hr growth curve are shown for the Δ<i>atpG</i> mutant as a percentage of the WT maximums. <b>C.</b> Minimum inhibitory concentrations were measured for <i>S</i>. <i>aureus</i> WT and Δ<i>atpG</i> in TSB 5g/L glucose and are shown as a ratio to the WT MIC (n = 3).</p