Michael P. Spector

Pushing the envelope: extracytoplasmic stress responses in bacterial pathogens

Despite being nutrient rich, the tissues and fluids of vertebrates are hostile to microorganisms, and most bacteria that attempt to take advantage of this environment are rapidly eliminated by host defences. Pathogens have evolved various means to promote their survival in host tissues, including stress responses that enable bacteria to sense and adapt to adverse conditions. Many different stress responses have been described, some of which are responsive to one or a small number of cues, whereas others are activated by a broad range of insults. The surface layers of pathogenic bacteria directly interface with the host and can bear the brunt of the attack by the host armoury. Several stress systems that respond to perturbations in the microbial cell outside of the cytoplasm have been described and are known collectively as extracytoplasmic or envelope stress responses (ESRs). Here, we review the role of the ESRs in the pathogenesis of Gram-negative bacterial pathogens.

Multiple Factors Independently Regulate hilA and Invasion Gene Expression in Salmonella enterica Serovar Typhimurium

HilA activates the expression of Salmonella enterica serovar Typhimurium invasion genes. To learn more about regulation of hilA, we isolated Tn5 mutants exhibiting reduced hilA and/or invasion gene expression. In addition to expected mutations, we identified Tn5 insertions in pstS, fadD, flhD, flhC, and fliA. Analysis of the pstS mutant indicates that hilA and invasion genes are repressed by the response regulator PhoB in the absence of the Pst high-affinity inorganic phosphate uptake system. This system is required for negative control of the PhoR-PhoB two-component regulatory system, suggesting that hilA expression may be repressed by PhoR-PhoB under low extracellular inorganic phosphate conditions. FadD is required for uptake and degradation of long-chain fatty acids, and our analysis of the fadD mutant indicates that hilA is regulated by a FadD-dependent, FadR-independent mechanism. Thus, fatty acid derivatives may act as intracellular signals to regulate hilA expression. flhDC and fliA encode transcription factors required for flagellum production, motility, and chemotaxis. Complementation studies with flhC and fliA mutants indicate that FliZ, which is encoded in an operon with fliA, activates expression of hilA, linking regulation of hilA with motility. Finally, epistasis tests showed that PhoB, FadD, FliZ, SirA, and EnvZ act independently to regulate hilA expression and invasion. In summary, our screen has identified several distinct pathways that can modulate S. enterica serovar Typhimuriums ability to express hilA and invade host cells. Integration of signals from these different pathways may help restrict invasion gene expression during infection.

The Starvation-Stress Response (SSR) of Salmonella

Salmonella serovars are common etiologic agents of intestinal-based disease of animals and humans. As a result of their lifestyle, salmonellae occupy and survive in a wide range of niches where they can encounter an even broader range of environmental stresses. One of the most common stresses is starvation for an essential nutrient such as a carbon/energy (C)-source. The genetic and physiologic changes that the bacterium undergoes in response to starvation-stress are referred to as the starvation-stress response or SSR. The genetic loci whose expression increases in response to the starvation-stress compose the SSR stimulon. Several loci of the SSR stimulon have been identified in Salmonella typhimurium and grouped, based on putative or known functions or products, into transport systems, C-compound catabolic enzymes, known protective enzymes, respiratory enzyme systems, regulatory proteins, virulence loci and unclassified products. The majority of loci identified are under positive control by the rpoS-encoded sigma factor, sigma S. However, a few are under (indirect) negative control by sigma S, but only during starvation-induced stationary phase. Most of the loci identified are also under either positive or negative control by the cAMP:CRP complex. For many, additional regulatory proteins (e.g. FadR, OxyR, and RelA and others) play a role in their regulation as well. Furthermore, most of the SSR loci identified are induced during other stresses or environmental conditions. For example, some are induced during P- or N-starvation, in addition to C-starvation; some are induced by extremes in pH or osmolarity; and some are induced in the intracellular environment of epithelial cells, and/or macrophages, and/or medium designed to mimic the intracellular milieu of mammalian cells (ISM). Several SSR loci are required for long-term starvation-survival (core SSR loci), e.g. narZ, dadA, stiC and rpoS. In addition, a few of the core SSR loci are also required for stress-specific-inducible and/or C-starvation-inducible resistance to H2O2 (e.g. stiC), thermal (e.g. stiC), and/or acid pH (e.g. narZ), challenge. Interestingly, C-starved cells are resistant to challenge with the antimicrobial peptide, polymyxin B. However, this resistance mechanism(s) is different from the resistance mechanisms for H2O2 and other environmental stresses. Furthermore, a link between the SSR and Salmonella virulence can be hypothesized since the two major regulators of the SSR, sigma s and cAMP:CRP, are required for full virulence of Salmonella. Moreover, the spv (Salmonella plasmid-associated virulence) genes, required for Salmonella to cause systemic disease, are C (and P- and N-)-starvation-inducible. However, a direct link between starvation-stress and virulence has not been established conclusively.

The rpoS-dependent starvation-stress response locus stiA encodes a nitrate reductase (narZYWV) required for carbon-starvation-inducible thermotolerance and acid tolerance in Salmonella typhimurium

The starvation-stress response (SSR) of Salmonella typhimurium includes gene products necessary for starvation avoidance, starvation survival and virulence for this bacterium. Numerous genetic loci induced during carbon-source starvation and required for the long-term-starvation survival of this bacterium have been identified. The SSR not only protects the cell against the adverse effects of long-term starvation but also provides cross-resistance to other environmental stresses, e.g. thermal challenge (55 degrees C) or acid-pH challenge (pH 2.8). One carbon-starvation-inducible lac fusion, designated stiA was previously reported to be a sigma(S)-dependent SSR locus that is phosphate-starvation, nitrogen-starvation and H2O2 inducible, positively regulated by (p)ppGpp in a relA-dependent manner, and negatively regulated by cAMP:cAMP receptor protein complex and OxyR. We have discovered through sequence analysis and subsequent biochemical analysis that the stiA::lac fusion, and a similarly regulated lac fusion designated sti-99, lie at separate sites within the first gene (narZ) of an operon encoding a cryptic nitrate reductase (narZYWV) of unknown physiological function. In this study, it was demonstrated that narZ was negatively regulated by the global regulator Fnr during anaerobiosis. Interestingly, narZ(YWV) was required for carbon-starvation-inducible thermotolerance and acid tolerance. In addition, narZ expression was induced approximately 20-fold intracellularly in Madin-Darby canine kidney epithelial cells and 16-fold in intracellular salts medium, which is believed to mimic the intracellular milieu. Also, a narZ1 knock-out mutation increased the LD50 approximately 10-fold for S. typhimurium SL1344 delivered orally in the mouse virulence model. Thus, the previously believed cryptic and constitutive narZYWV operon is in fact highly regulated by a complex network of environmental-stress signals and global regulatory functions, indicating a central role in the physiology of starved and stressed cells.

Starvation-inducible loci of Salmonella typhimurium : regulation and roles in starvation-survival

Four starvation‐inducible loci (stiA, stiB, stiC, and stiE) of Salmonella typhimurium have been extensively characterized as to their genetic and physiologic regulation, and their roles in survival during prolonged simultaneous phosphate (P)‐, carbon (C)‐and nitrogen (N)‐starvation (PCN‐starvation). Strains of S. typhimurium LT‐2, isogenic with the exception of lacking either the stiA, stiB or stiC locus, died off more quickly and survived at much reduced levels compared with their wild‐type parent. When certain sti mutations were combined in the same strain, we found that viability of these cultures declined even more rapidly, and starvation‐survival was affected to leveis over‐and‐above the additive effects of each individual mutation, indicating an epistatic relationship between these loci. All four sti loci were, directly or indirectly, under negative control by the crp gene product (cAMP receptor protein, CRP). With the exception of stiB, all were similarly regulated by the cya gene product (i.e., cAMP). This suggests that CRP acts alone, or with a signal molecule other than cAMP, to cause repression of the stiB locus. In addition, all four loci are under positive regulation by the relA gene product (i.e., ppGpp) during C‐ or N‐starvation, but not P‐starvation. Since not all relA‐depen‐dent sti loci are induced during both C‐ and N‐starvation, we propose that two separate ppGpp‐dependent pathways function during C‐starvation and N‐starvation, respectively. Possible models for separate P‐, C‐and N‐starvation‐induction pathways are discussed.

Essential roles of core starvation‐stress response loci in carbon‐starvation‐inducible cross‐resistance and hydrogen peroxide‐inducible adaptive resistance to oxidative challenge in Salmonella typhimurium

The starvation‐stress response (SSR) of Salmonella typhimurium encompasses the physiological changes that occur upon starvation for an essential nutrient, e.g. C‐source. A subset of SSR genes, known as core SSR genes, are required for the long‐term starvation survival of the bacteria. Four core SSR loci have been identified in S. typhimuriumrpoSstiAstiB, and stiC. Here we report that in S. typhimurium C‐starvation induced a greater and more sustainable cross‐resistance to oxidative challenge (15 mM hydrogen peroxide (H2O2) for 40 min) than either N‐ or P‐starvation. Of the four core SSR loci, only rpoS and stiC mutants exhibited a defective C‐starvation‐inducible cross‐resistance to H2O2 challenge. Interestingly, (unadapted) log‐phase S. typhimurium rpoS and stiA mutants were very sensitive to oxidative challenge. Based on this, we determined if these core SSR loci were important for H2O2 resistance developed during a 60 min adaptive exposure to 60 μM H2O2 (adapted cells). Both unadapted and adapted rpoS and stiA mutants were hypersensitive to a H2O2 challenge. In addition, a stiB mutant exhibited normal adaptive resistance for the first 20 mins of H2O2 challenge but then rapidly lost viability, declining to a level of about 1.5% of the wild‐type strain. The results of these experiments indicate that: (i) the rpoS and stiC loci are essential for the development of C‐starvation‐inducible cross‐resistance to oxidative challenge, and (ii) the rpoSstiA, and, in a delayed effect, stiB loci are needed for H2O2‐inducible adaptive resistance to oxidative challenge. Moreover, we found that both stiA and stiB are induced by a 60 μM H2O2 exposure, but only stiA was regulated (repressed) by (reduced form) OxyR.

Role of Periplasmic Peptidylprolyl Isomerases in Salmonella enterica Serovar Typhimurium Virulence

ABSTRACT FkpA is a peptidylprolyl isomerase whose expression is regulated by the alternative sigma factor, sigma factor E (σE). In contrast to the results of a previous report, inactivation of fkpA was found to have only a minor effect on the ability of Salmonella enterica serovar Typhimurium to invade and survive within epithelial and macrophage cell lines and cause infection in mice. However, an effect of the fkpA mutation on serovar Typhimurium virulence was seen if the mutation was combined with mutations in surA or htrA, two other σE-regulated genes, which encode proteins involved in protein folding and/or degradation in the periplasm.

Regulation of NAD Biosynthesis in Salmonella typhimurium: Expression of nad—lac Gene Fusions and Identification of a nad Regulatory Locus

Regulation of NAD biosynthesis was examined through the construction of nad-lac fusions in Salmonella typhimurium. The nadA (17 unit map position) and nadB (55 units) genetic loci involved with quinolinic acid biosynthesis were both found to be regulated by the product of a nadR locus (99 units) in a repression/derepression manner while nadC (3 units) expression appeared constitutive at the transcriptional level. Increases in nadAB transcription directly correlated with decreases in intracellular NAD(P) levels, and kinetic studies indicated that the NAD analogue 6-amino NAD was ineffective in repressing either nadA or nadB. The presence of cAMP + cAMP receptor protein was essential for the complete derepression of nadA while no effect was evident upon nadB. Transfer of cultures from aerobic to anaerobic conditions, however, resulted in the partial derepression of both nadA and nadB. Thus, there appears to be a very complex set of controls regulating NAD biosynthesis.

Genetic characterization of pyridine nucleotide uptake mutants of Salmonella typhimurium.

Two classes of pyridine nucleotide uptake mutants isolated previously in a strain of Salmonella typhimurium defective in both de novo NAD biosynthesis (nad) and pyridine nucleotide recycling (pncA) were analysed in terms of their genetic relationship to each other and their roles in the transport of nicotinamide mononucleotide as a precursor to NAD. The first class of uptake mutants, pnuA (99 units), failed to grow on nicotinamide mononucleotide (NMN) as a precursor for NAD. The second class, pnuB, grew on lower than normal levels of NMN and suppressed pnuA mutations. A third class of uptake mutant, pnuC, isolated in a nadB pncA pnuB background, also failed to grow on NMN. Transport studies and enzyme analyses confirmed these strains as defective in NMN uptake. A fourth locus, designated pnuD, was found to diminish NMN utilization in a nad pncA+ background. Tn10 insertions near pnuA, pnuC and pnuD were isolated and utilized in mapping studies. pnuA was found to map between thr and serB near trpR. The pnuC locus was cotransducible with nadA at 17 units while pnuD mapped at approximately 60 units. The biochemical and genetic data suggest that the pnuA and pnuC gene products cooperate in the utilization of NMN under normal conditions. A pnuB mutant, however, does not require the pnuA gene product for NMN uptake but does rely on the pnuC product. Fusion studies indicate that pnuC is regulated by internal NAD concentrations.

Starvation-Stress Response (SSR) of Salmonella typhimurium

The environments that many pathogenic bacteria encounter as they cycle from their host organism(s) to the external aquatic or terrestrial environments commonly share one general characteristic: they are frequently limiting for bacterial growth (Koch, 1971). A wide variety of environmental factors can play an important role in restricting bacterial growth, e.g., pH, osmolarity, and the availabilities of oxygen as well as essential nutrients (Tempest et al., 1983; Roszak and Colwell, 1987). The availability of essential nutrients such as phosphate (P), carbon (C), or nitrogen (N) in these environments is of particular importance in limiting bacterial growth (Harder and Dijkhuizen, 1983). As a consequence, non-spore-forming bacteria, e.g., Salmonella, frequently undergo drastic metabolic readjustments in an effort to survive until more favorable conditions are encountered.