Regulation of Bacterial Virulence

This chapter focuses on the relationships among biofilm development, the environment, and antimicrobial tolerance for the paradigm organism Pseudomonas aeruginosa. While a specific strain of P. aeruginosa consistently produces the same biofilm structure under one laboratory culturing condition, the same strain produces a very different biofilm structure under different culturing conditions. Investigators have discovered that biofilms grown in vitro reproducibly form specific structures that are affected by a plethora of conditions. For P. aeruginosa, two general biofilm shapes have been observed using the flow cell system: structured biofilms and flat biofilms. While the environmental sensing mechanisms and the regulatory pathways leading to the formation of specific biofilm structures have not been fully elucidated, it is clear that many factors are important for this process. A focus of recent work has been on identifying the signal transduction and regulatory pathways that control biofilm formation and that integrate different environmental signals during biofilm development. The chapter provides an outline about the regulation of biofilm formation by cyclic-di-GMP (c-di-GMP) and two-component systems (TCSs). Researchers are currently trying to determine the nature of these environmental signals, and as they do, our understanding of which environments promote or impair biofilm formation will grow. Microbial fuel cells and wastewater treatment communities are a few examples of engineered, structured communities that could benefit from such an approach.

This chapter reviews the observations supporting the idea that acute and chronic Pseudomonas aeruginosa infections represent distinct modes of host-pathogen interaction. The virulence factors associated with acute infections and chronic infections are discussed, with a focus on data obtained from human subject-based studies, when possible. P. aeruginosa expresses many virulence factors that can damage host cells and which contribute to infection in both humans and animal models. Ectopic expression of virulence factor regulator (Vfr) in mucA22 strains restored expression of ExoS, type IV pilus (TFP), and elastase, confirming that downregulation of Vfr is responsible for decreased virulence factor expression in mucoid strains under the conditions evaluated in this study. While it is clear that regulators of TFP biogenesis and function are intimately associated with the control of Vfr and cyclic AMP (cAMP) expression, the mechanism that links twitching motility and Vfr remains to be elucidated. rsmZ and rsmY are two sRNAs that interact with the RNA binding protein RsmA. The paper by Bordi and coworkers also provides the first data that rsmY and rsmZ are not functionally redundant. Increased expression of T6SS genes, whose translation is negatively regulated by RsmA, specifically requires increased expression of the rsmZ sRNA. Most bacterial pathogens, P. aeruginosa included, use their virulence factors for the primary purpose of gaining access to nutrients rather than for causing damage to specific hosts. The chapter focuses on a few examples of regulatory networks that broadly impact virulence factor expression.

This chapter focuses on quorum sensing in Burkholderia, specifically Burkholderia pseudomallei, Burkholderia thailandensis, and Burkholderia mallei (the Bptm group). This group has highly conserved quorum sensing systems, yet each species occupies strikingly different environments. Quorum sensing systems have been found in all of the Burkholderia species studied to date. Prior to discussing the quorum sensing components of the Bptm group, it is important to understand the evolutionary history and lifestyle of each species. B. thailandensis and B. pseudomallei are saprophytic bacteria found in the soil and water in tropical regions common to Southeast Asia, northern Australia, South America, the Middle East, and some regions in Africa. Diagnosis and treatment of melioidosis are challenging because the disease presents with various symptoms and B. pseudomallei is intrinsically multidrug-resistant. Quorum sensing was first described to occur in the Bptm group within the past decade. The quorum sensing circuits in these bacteria are among the most complex acylated homoserine lactone (AHL) systems described. A summary of the quorum sensing components and AHL signals for each species is discussed in the chapter. Quorum sensing in B. pseudomallei has many parallels with quorum sensing in P. aeruginosa. Bacterial adherence, aggregation into microcolonies, and biofilm formation are important survival factors during the saprophytic and host-associated lifestyle of many opportunistic pathogens. The quorum sensing-controlled phenotypes observed in B. thailandensis are consistent with the idea that this bacterium uses quorum sensing during its saprophytic lifestyle.

Staphylococcus aureus is a gram-positive bacterium that is a component of the commensal flora of the skin and the nares. S. aureus is responsible for causing significant morbidity and mortality, resulting in nearly 300,000 hospitalizations and 19,000 deaths in the United States annually. Surface-associated factors enable S. aureus to bind to biotic surfaces, abiotic surfaces, and itself. The ability to adhere to varied surfaces likely represents the first step during the infection process, in which the bacterium attaches and grows on different tissues. The ability of S. aureus to infect many tissues in the mammalian body suggests that this bacterium is extremely efficient at adapting to different environments. In order to monitor the density of the population and prevent starvation and/or clearance by the host, most bacteria secrete small molecules into the extracellular milieu that accumulate in response to an increase in the number of bacteria in a defined space. These so-called autoinducers are sensed by the entire population, triggering a signaling cascade that informs the community that quorum has been attained. Numerous regulatory networks work with or against one another to carefully coordinate the precise expression and production of a large collection of virulence factors that play different roles in S. aureus infection. A critical layer of complexity to this topic is the tremendous strain-to-strain variability seen in clinical isolates. This variability often influences the expression of virulence factors, which directly alters the pathogenic trait of clinical isolates.

This chapter discusses the mechanisms by which vertebrates sequester iron from invading pathogens and the response of pathogens to this sequestration. It provides examples of iron-regulated virulence determinants in several clinically important gram-positive bacteria. Iron is crucial to the activity of ribonucleotide reductase, nitrogenase, peroxidase, catalase, and succinic dehydrogenase, and it is therefore required for the vital functions of respiration and several metabolic pathways. During infection, pathogens must rely on their host as the sole source of nutrient iron. Diseases in iron metabolism impact susceptibility to infection, exemplified by an increased frequency of infections caused by Salmonella, Mycobacterium, and Plasmodium in patients with high iron levels. Transcriptional regulation of bacterial genes in response to iron occurs through the activity of metal-dependent regulators. Corynebacterium diphtheriae is the causative agent of diphtheria, a contagious upper respiratory tract infection that has been largely eradicated in the last century due to worldwide utilization of the diphtheria vaccines. The iron-containing tetrapyrrole heme is the preferentially bound iron source of Staphylococcus aureus. Iron-dependent virulence gene expression in S. aureus involves a complex regulatory network comprised of Fur and the two-component systems Agr and Sae, which regulate quorum sensing and secreted virulence factors, respectively.

This chapter covers the role of iron regulation in the virulence of gram-negative bacterial pathogens, where possible, using Yersinia pestis as a paradigm. In most gram-negative bacteria, including Y. pestis, the alterations in mRNA and protein levels in response to iron availability are due to inherent iron requirements and iron homeostasis mechanisms controlled by two primary regulatory systems: the iron (Fe) uptake regulation protein Fur and the small RNA (sRNA) RyhB or its analogs (e.g., PrrF and NrrF). The chapter focuses on these two regulators, their regulons, and their roles in virulence. While Fur and RyhB regulons are often described separately, they are in fact interrelated regulatory networks. In a variation on the mRNA degradation theme for sodB, RyhB selectively promotes degradation of only the 3' portion of the iscRSUA mRNA, while preserving expression of IscR, a transcription factor encoded by the first gene of the operon. The chapter then focuses on the iron transport systems of Y. pestis. A section on RyhB regulatory mechanisms explains that RyhB regulation increases the pools of both shikimate and serine, while indirectly contributing to expression of the enterobactin biosynthesis genes. The mechanisms of Fur activation, Mn-Fur regulation, and apo-Fur regulation as well as the different regulatory mechanisms of RyhB are addressed. However, the complexity of iron regulation mechanisms and their role in microbial pathogenesis will likely provide interesting questions to resolve for decades to come.

This chapter describes the virulence and regulation of chaperone-usher pathway (CUP) pili, focusing on type 1 and P pili in uropathogenic Escherichia coli (UPEC), the most common cause of urinary tract infections (UTIs). UPEC and E. coli in general heavily rely on CUP pili to mediate attachment to biotic and abiotic surfaces. The pathogenesis of community-acquired UPEC UTI is thought to begin with UPEC colonization of the periurethral area from the fecal flora. Depending on the specific niche UPEC inhabits, it encounters different elements of the immune response as well as different environmental pressures, necessitating precise gene regulation. The ability of UPEC to transcend the acute population bottleneck is described in detail. In addition to up-regulation of genes involved in general metabolism, several genes classified as being involved in removal of reactive oxygen species and hydrophobic compounds were also upregulated. Further, negative cross-regulatory interactions between pili may serve to divert resources to the conditions most effective for persistence or transit throughout hosts. Antivirulence compounds such as pilicides and mannosides represent novel strategies to translate basic knowledge from the investigation of pilus structure and function into new therapeutics that may have efficacy in treating UTIs by affecting CUP expression and function.

This chapter discusses the molecular mechanisms of phase variation and the possible roles of phase variable restriction-modification (R-M) systems in bacterial pathogens and reveals how a number of phase-variable type III R-M systems have evolved to have a new and distinct function in gene regulation that results in generation of a diverse bacterial population. Phase variation via simple tandem repeats is by far the most common mechanism of phase variation. Phase variation mediated by DNA methylation is different from the mechanisms. While these mechanisms result from changes in the genome, DNA methylation is epigenetic, meaning that while the phenotype differs the DNA sequence remains unaltered. The fundamental characteristic of the DNA methylation-dependent phase-variable systems is that the methylation state of the target site affects the DNA binding of a regulatory protein, which directly regulates transcription. Importantly, a distinct mod is associated with a hypervirulent clonal lineage of meningococci, and its phasevarion includes genes suggested to be virulence factors. The presence of multiple phase-variable mod alleles suggests the possibility of distinct phasevarions existing within each strain, each regulating a different set of genes. The chapter proposes that the phase-variable methylation has arisen due to the selective advantage conferred by the phase-varion enabling random switching of an organism between two distinct cell types. In organisms with multiple phasevarions switching independently, multiple differentiated cell types can be generated.

Alginate is arguably the best-characterized exopolysaccharide produced by Pseudomonas aeruginosa, and several excellent reviews have been written on the molecular biology of its production and clinical ramifications. This chapter reviews the transcriptional factor and posttranscriptional factor involved in controlling and inducing alginate production. The gene encoding the cognate histidine kinase for the response regulator AlgB is kinB (PA5484 on the PAO1 chromosome), located directly downstream of algB on the PAO1 chromosome. Deletion of mucR also affected other known cyclic dimeric-GMP (c-di-GMP) processes in P. aeruginosa, including swarming motility, biofilm formation, and alginate production. It additionally showed, through the use of lacZ and phoA fusions, that the predicted amino-terminal MHYT domain of MucR resides on the inner membrane. MHYT domains are proposed to bind O2, NO, or CO. A model for c-di-GMP regulation of alginate production was proposed whereby the guanylate cyclase of MucR is stimulated by a yet-to-be-identified signal, which binds to a predicted MHYT domain in the amino terminus of MucR. The current state of knowledge indicates that Psl and Pel are likely involved with the initial stages of biofilm development, whereas alginate is the stress response exopolysaccharide. The levels of alginate produced by newly generated MucA, MucB, MucC, or MucD mutants of P. aeruginosa PAO1 and other nonmucoid clinical isolates (i.e., non-CF isolates) was found to be inversely related to biologically relevant concentrations (e.g., <5 to 100 μM) of iron present in the media used in this study.

Vaccination represents the best prospect for managing pneumococcal disease in the 21st century. Polyvalent purified capsular polysaccharide (CPS) vaccines introduced in the 1980s confer strictly serotype-specific protection and are poorly immunogenic in young children. Consequently, current global efforts are now focused on accelerating the development of alternative pneumococcal vaccines based on proteins that contribute to pathogenesis and are common to all serotypes. Bioinformatic analysis suggests that catabolite control protein A (CcpA) can potentially regulate expression of other pneumococcal surface proteins, including StrH (an N-acetylglucosaminidase), GlpO (alpha-glycerophosphate oxidase), and MalX (a maltose/maltodextrin ABC transporter). The majority of CPS serotypes are highly charged at physiological pH, and electrostatic repulsion may directly interfere with interactions with phagocytes. The prospect of developing vaccines targeted at pneumococcal surface proteins increases the importance of understanding their role in pathogenesis, their relative expression levels in various host compartments, and the mechanism(s) whereby their expression in vivo is regulated. Current knowledge on regulatory mechanisms operating on various classes of pneumococcal surface proteins is provided in this chapter. Streptococcus pneumoniae is a highly successful, human-adapted pathogen, responsible for more than a million deaths each year. The complexity of these regulatory networks makes the task of identifying the principal determinants of virulence gene expression a challenging one. Nevertheless, a thorough dissection of the critical regulatory pathways employed by S. pneumoniae in discrete in vivo niches will undoubtedly provide an improved understanding of pneumococcal pathogenesis and possibly identify novel targets for intervention.

Lipopolysaccharide (LPS) is the major component of the outer membrane of gram-negative bacteria and consists of three distinct structural domains: lipid A, a nonrepeating “core” oligosaccharide, and a distal repeated O-antigen polysaccharide. This chapter discusses the structure of the three regions, in order, as they extend out from the outer membrane, focusing on regulated alterations, modifications, and/or substitutions. The biosynthetic enzymes are either constitutively active or regulated by a variety of two-component regulatory systems, including PhoR/PhoB, PmrA/PmrB and/or PhoP/PhoQ. Deletion of IpxT in Escherichia coli resulted in an increase in sensitivity to the cationic antimicrobial peptides (AMPs) polymyxin B, although experiments elucidating roles for lpxT in overall pathogenesis have yet to be undertaken. The chapter explores the regulation of one particular modification, that of phosphorylcholine (ChoP), on Haemophilus and Neisseria lipooligosaccharide (LOS). Regulation of LPS core biosynthesis is not well understood, although some of the regulatory mechanisms for biosynthesis of Kdo and inner and outer core are emerging. The current evidence based on genetic and biophysical interaction studies of the Lpt proteins supports the transenvelope model. Further studies into the regulation of LPS should help provide a link between signals in the environment and the resulting outer membrane composition that are likely to have the most impact on host-pathogen interactions.

Of the more than 200 different serogroups of Vibrio cholerae that have been isolated, only two of these, O1 and O139, have been found to have epidemic and pandemic potential. CTXφ carries the genes for CT (ctxA and ctxB) and the VPI contains the genes (tcpA-E and tcpJ) responsible for the synthesis and assembly of the essential colonization factor toxin-coregulated pilus (TCP). As part of the acetoin operon, AphA represses the expression of two PhoB-activated genes, acgA and acgB, that encode proteins which influence motility and biofilm formation by altering c-di-GMP levels in the cell. Bicarbonate may be an important in vivo signal that increases the activity of ToxT during infection and induces virulence gene expression. Once V. cholerae has disseminated out of the host and virulence gene expression is no longer required, TcpP and ToxT levels are decreased by proteolysis and H-NS reestablishes repression at the various promoters. Thus, like GbpA, FrhA plays an important role in V. cholerae in both the host and in the aquatic environment. A model for V. cholerae infection involves motile bacteria attaching to the intestinal cell surface, after which they upregulate virulence factor expression and downregulate motility. The flagellar regulatory hierarchy also influences virulence gene expression in V. cholerae through the quorum sensing system. Recent advances in the development of cDNA sequencing (RNA-seq) have facilitated the generation of comprehensive transcriptome profiles of V. cholerae during infection in both the rabbit and mouse models of cholera.

The best-studied members of the Bacillus cereus group, B. anthracis, B. thuringiensis, and B. cereus sensu stricto, are pathogens with common and unique features that facilitate their ability to cause disease. As the etiological agent of anthrax, B. anthracis is the most renowned member of the B. cereus group. Inhalation or ingestion of B. anthracis spores can result in a lethal hemorrhagic septicemia. Anthrax toxin represents an interesting variation on the classic A-B toxin model: one binding/translocating B component, protective antigen (PA), and two enzymatic A components, edema factor (EF) and lethal factor (LF). Opportunistic infections caused by B. thuringiensis and B. cereus sensu stricto are relatively uncommon, but they can have serious consequences whether local or systemic. The chromosomes of B. anthracis, B. thuringiensis, and B. cereus sensu stricto reveal striking sequence similarity and gene synteny, but virulence-associated plasmid content can allow facile discrimination of the three species. A large number of virulence factors have been established for the pathogenic B. cereus group species. Anthrax toxin is the best-studied and arguably the most important virulence factor produced by B. anthracis. The entomopathogenesis of B. thuringiensis is dependent upon the production of characteristic insecticidal parasporal crystals called cryotoxins (Cry) and cytolysins (Cyt). B. anthracis, B. thuringiensis, and B. cereus senso stricto secrete pore-forming toxins of the cholesterol-dependent cytolysin (CDC) family.

Clostridium perfringens is a gram-positive, spore-forming anaerobic rod that is widespread in the environment and is commonly isolated from the gastrointestinal tract of humans and animals, as well as from soil and sewage. This chapter provides an overview of the regulatory systems and mechanisms involved in the control of toxin production in C. perfringens. Two-component signal transduction systems represent one of the most widespread mechanisms by which bacteria sense and respond to a diverse range of changes in both environmental stimuli and bacterial cell density. Genetic studies have shown that disruption of either virR or virS resulted in an altered toxin production profile. Although VirSR was first identified as a positive regulator of extracellular toxin production, it is now considered a bifunctional system, as it has been demonstrated to positively and negatively regulate the expression of many genes at the transcriptional level. The maintenance of the correct helical phasing, the correct spacing between the VirR boxes, and the correct distance between the VirR boxes and the -35 region were shown to be critical for optimal transcriptional activation. Genomic analysis has predicted a number of small regulatory RNAs (sRNAs) in the genomes of C. perfringens isolates. In the C. perfringens gas gangrene strains, 13 and ATCC 13124, 193 and 181 sRNAs have been predicted, respectively, whereas 131 sRNA were predicted in the food poisoning isolate, SM101.

This chapter discusses our current understanding of gene regulation in Clostridium difficile, focusing on how toxin production is regulated, with a particular emphasis on the major toxins, toxin A and toxin B. The onset of toxin synthesis is associated with entry into the stationary phase of growth. The precise growth phase signals involved in the initiation of toxin production remain unknown, even though nutrient signals have clearly been shown to have a profound effect on toxin production by C. difficile. TcdA and TcdB are encoded by tcdA and tcdB, respectively, which are located in a region of the chromosome known as the pathogenicity locus (PaLoc). The TcdR protein has a helix-turn-helix DNA binding motif and has limited similarity to some clostridial transcriptional activator proteins, as well as to several families of eubacterial RNA polymerase sigma factors, which is consistent with the hypothesis that TcdR is a positive regulator of toxin production. The regulation of toxin production in C. difficile is clearly complex and relies on a number of different regulatory systems. In Bacillus species, activation of Spo0A occurs via a phosphorelay cascade that eventuates in the phosphorylation of Spo0A via Spo0F and Spo0B. SigH is an alternative sigma factor that is involved in the transition to stationary phase and sporulation. Recent breakthroughs in genetic manipulation technologies available for C. difficile, together with refined animal infection models, will facilitate future studies and will further define the complex regulatory networks involved in C. difficile toxin regulation.

In order to cause disease, pathogenic bacteria require specialized means to sense the various microenvironments presented to them in the context of a host and then to regulate the systems required for establishment, persistence, growth, and induction of the pathologies associated with infection. The example presented in this chapter focuses on one such system utilized by Bacillus anthracis during anthrax infections, iron acquisition. Recently, research focused on the acquisition of one such nutrient essential for successful anthrax infections, iron, has come to the fore. The chapter summarizes the various mechanisms used by B. anthracis for obtaining iron, reviews the relative impact of each of these mechanisms on a successful anthrax infection, and presents the transcriptome regulated by low concentrations of iron. Once inside the bacterial cytoplasm, iron is released from heme when the heme monooxygenase, IsdG, breaks down the molecule. Siderophores are high-affinity iron-chelating molecules that are secreted into the extracellular environment, where they scavenge iron from a variety of host sources. B. anthracis produces two siderophores, bacillibactin and petrobactin. Petrobactin is made up of one central citrate flanked by two spermidine molecules, each with terminal dihydroxybenzoic acid residues. Consequently, molecules that inhibit or block iron acquisition, related membrane transport processes, or bacterial iron-based regulation, in general, may prove to be effective new medical counter-measures against anthrax and related infections. It is predicted that as more defined details of the genes, proteins, factors, and mechanisms that regulate iron metabolism become understood, reasonable countermeasure targets will be identified.

This chapter presents a general overview of type III secretion system (T3SS) regulation in the main pathogenic bacteria, and focuses on the different aspects of the regulation of T3SS gene expression in Pseudomonas aeruginosa. Pathogenic bacteria occupy very different infection foci; for instance, the animal pathogens Shigella spp. and Salmonella spp. live intracellularly after successful invasion, whereas Yersinia spp., P. aeruginosa, and enteropathogenic Escherichia coli (EPEC) predominantly remain extracellular. Therefore, different stimuli could be used to up or downregulate the expression of T3SS genes: temperature, divalent cations, host cell contact, serum, or other factors. Although the mechanism by which metabolic pathways influence virulence gene expression in bacteria is unclear, the example of P. aeruginosa detailed could facilitate progress in that field. It has been shown that T3SS expression is dependent on cell density and that exsCEBA operon expression decreases rapidly in the second part of stationary phase, and also that indole-3-acetic acid (IAA), naphthalenacetic, and 3-hydroxykynurenine inhibit exsCEBA operon expression at millimolar concentrations. In 2005, Wu and coworkers proposed that the opportunistic pathogen P. aeruginosa could adapt to the host by sensing alterations in the host immune function and respond by enhancing the virulence phenotype. Despite the large number of genes that influence the regulation of the expression of T3SS genes in P. aeruginosa, only a minority have been shown to have a precise role. Both fundamental and applied research can make the inhibition of T3SS expression as one of the first new antibacterial therapeutics.

This chapter explores the question of how bacterial pathogens regulate the biogenesis and function of their type IV secretion systems (T4SSs) in pathogenic settings. First, it describes a regulatory cascade involving the perception of multiple signals exchanged between Agrobacterium tumefaciens and its plant host. This signaling dialogue leads not only to infection of plant tissue but also to enhanced conjugative transfer of the virulence-associated tumor-inducing (pTi) plasmid. Second, it summarizes the regulatory features of a large T4SS subfamily, the conjugation systems functioning in gram-negative and -positive species. The chapter then summarizes regulatory features of the well-characterized effector translocators of Brucella spp., Legionella pneumophila, and Bartonella spp., and also examines why and how these and other bacterial pathogens cross-regulate T4SSs and other surface motility or attachment devices such as flagella and type IV pili. Finally, the chapter discusses post-transcriptional regulation of substrate-T4SS docking reactions and donor-target cell contacts. The overarching goal of this chapter is to identify mechanistic themes and variations that have evolved to regulate the myriad of T4SS activities exploited by bacterial pathogens during infection. The conjugation systems are the largest subfamily, present in nearly all bacterial species and some archaeal species. Many bacterial pathogens rely on flagellar or type IV pilus-based motility to migrate to sites favorable for colonization within the host. For all T4SSs, transduction of exogenous or physiological signals ultimately converges on the regulatory machinery controlling transcription of machine subunits, DNA processing enzymes, or protein effectors.

The facultative intracellular bacterium Listeria monocytogenes has been used for decades as a model infectious agent for the study of host innate and adaptive immunity. This chapter focuses on describing what is currently known about how L. monocytogenes mediates the transition from saprophyte to mammalian pathogen and explores the regulatory circuit governed by a transcriptional activator known as PrfA that allows the bacterium to flourish in both soil and cytosol. In healthy individuals, disease caused by L. monocytogenes is usually self-limiting and presents as a form of mild gastroenteritis. The primary route of L. monocytogenes infection is translocation across the intestinal epithelium following the consumption of contaminated food products. The correct compartmentalization of broad range phospholipase C (PC-PLC) activity is important for avoiding damage to host cell membranes and is a critical aspect of L. monocytogenes virulence. L. monocytogenes strains containing prfA* mutations are hyperinvasive, are quicker to mediate phagosome escape, and initiate bacterial actin-based motility more rapidly to promote cell-to-cell spread. Microarray analyses of L. monocytogenes wild-type and prfA* mutants grown in brain heart infusion broth suggest the possibility of at least 145 or more additional genes associated with PrfA regulation. PrfA activation appears to function as the switch that enables L. monocytogenes to transition from saprophyte to pathogen. In contrast to carbon sources encountered within the host, PrfA-dependent gene expression is dramatically repressed in the presence of cellobiose and other readily metabolized sugars that are likely to be more prevalent in environments located outside of host cells.

This chapter focuses on the function of the regulatory system SsrAB of Salmonella enterica, which controls expression of virulence factors for the intracellular phase of the life of the pathogen. It discusses the experimental data and current understanding of the function of the two-component system SsrAB. SsrAB is encoded by genes in Salmonella pathogenicity islands 2 (SPI2) and is necessary to control expression of virulence genes during the intracellular stage of Salmonella infection. Intracellular survival and replication is dependent on the function of the SPI2-encoded T3SS (SPI2-T3SS). The signature-tagged mutagenesis screen initially identified various transposon-insertion mutants with highly reduced virulence in the murine model of systemic infection. A comparative analysis of expression levels under in vitro conditions indicated high diversity in expression levels of the various genes encoding structural components and effector proteins of the SPI2-T3SS. In contrast to the case with SsrA, several experimental approaches have been made towards understanding the function of SsrB, the response regulator of the SsrAB two-component system. The ancestral OmpR/EnvZ two-component system regulates the porin genes ompF and ompC in response to changes in osmolarity. The SPI1-encoded HilD regulatory protein, which activates the expression of SPI1 genes, has been suggested to control the switch from SPI1 to SPI2 induction. The levels of regulation range from the SPI2-encoded regulatory system SsrAB and core genome-encoded regulators to global control elements such as DNA topology regulators.

Francisella tularensis is a gram-negative coccobacillus that is found in diverse environments, including animal, protozoan, and insect hosts. Several animal models have been used for the study of F. tularensis infection, primarily rodents (including mice, rats, and guinea pigs) as well as rabbits. For all the obvious reasons, the majority of studies have been carried out in mouse models. The host response can be unique regarding F. tularensis infection. This can be largely due to the different in vivo environments this bacterial pathogen encounters during infection (e.g., temperature changes when F. tularensis is transmitted from arthropods to mammals). Production of reactive oxygen species and reactive nitrogen species is an essential innate immune defense mechanism against invading microorganisms. There are four known major regulators of virulence in F. tularensis: MglA, SspA, PmrA, and FevR, which positively control the expression of Francisella pathogenicity island (FPI) genes. Each of these proteins also regulates the expression of genes outside the FPI. Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Mutations in F. tularensis LVS capB and capC, which are similar to Bacillus anthracis capsule genes, had no effect on capsule expression.

This chapter presents the molecular mechanisms used by Salmonella to sense and respond to reactive species encountered at various phases during the infectious cycle. Salmonellae are exposed to reactive oxygen species (ROS) produced endogenously through the univalent or divalent reduction of O2 by enzymes of the electron transport chain or cytoplasmic flavoproteins. Oxyradicals generated by the NADPH phagocyte oxidase react with sulfur compounds in the gut lumen, generating the alternative electron acceptor tetrathionate. The effect of ROS on Salmonella central metabolism may be especially pertinent in phagosomes of macrophages, where nutrients might be a limited resource. The importance of thiol-mediated sensing of ROS and reactive nitrogen species (RNS) has been established in both prokaryotes and eukaryotes. Of interest to this chapter, SPI2 lessens the oxidative and nitrosative stress that Salmonella must endure within macrophages. A section briefly discusses the sources of NO and the chemistry of RNS relevant to Salmonella pathogenesis. The formation of dinitrosyliron complexes in fumarate/nitrate reduction (FNR) derepresses genes involved in the antinitrosative response of Salmonella. ROS and RNS have distinct biological chemistries, but they also share some common molecular targets. The realization that the SPI2 master regulator SsrB can be a sensor of RNS illustrates the complex strategies used by intracellular Salmonella to sense reactive species engendered in the course of the infection.

This chapter addresses the regulation of outer membrane vesicles (OMVs) production in gram-negative bacteria; however, a brief section is dedicated to summarizing current knowledge of gram-positive membrane vesicles (MVs). To understand the molecular mechanisms of OMV formation, it is important to first review the structural differences between the cell envelopes of Gram-negative and Gram-positive bacteria. Despite intense interest and research in the field since the discovery of OMVs, the molecular mechanism of OMV formation has not been completely elucidated. Three main models for the mechanism of OMV formation have been proposed, which are not mutually exclusive. Processes regulated by quorum sensing (QS) include production of secondary metabolites and virulence factors, light production, biofilm formation, and OMV formation. The contribution of OMVs to biofilm structures is discussed in the chapter; however, specifically within the host, the propensity to form microcolonies and the role OMVs play during infection may reveal novel biofilm-related regulatory mechanisms of OMV formation within the host. Through the combined efforts of many investigators over the course of decades of research, much light has been shed on the highly conserved process of bacterial MV formation, though several questions remain unanswered. Regulatory schemes for OMV formation are actively being determined, and some of the future progress could be derived from collaboration with other research areas like QS and regulatory RNAs.

This chapter discusses some of the stressors likely to target the cell envelope of Mycobacterium tuberculosis during infection, and the corresponding regulatory elements expressed by the bacterium to counteract this stress. Phylogenetically, M. tuberculosis is a member of the phylum Actinobacteria, which also includes several notable human pathogens, including species of the genera Streptomyces, Corynebacterium, Nocardia, and Rhodococcus. M. tuberculosis is a facultative intracellular pathogen, and its host range is restricted to humans. The bacterium is not normally found free within the environment, so its continued survival within the human population requires that it be transmitted directly from an infected individual with active disease to one that is susceptible to infection. Posttranslational phosphorylation of proteins was traditionally thought to be limited to eukaryotic cells. However, the discovery of two-component signal transduction systems (TCSSs) in bacteria shifted this paradigm to include phosphorylation of prokaryotic SKs on a conserved histidine residue and phosphorelay to a conserved aspartic acid residue on the cognate RRs. There is mounting evidence that PknB, and perhaps other STPKs, may also play an important regulatory role in the response of M. tuberculosis to environmental stress by directly regulating the activity of anti-sigma factors. A large body of work has helped to shape the current model of how M. tuberculosis senses cell envelope damage, including the regulatory mechanisms by which the bacterium responds to these stimuli.

Pathogenic bacteria express a myriad of systems aimed at subversion and exploitation of a host to promote proliferation and survival. Naturally, host cells have evolved equally complex defensive mechanisms which the bacterial pathogen must overcome in order to successfully establish an infection. Regulatory RNAs can operate at all layers of gene regulation, ranging from transcriptional initiation to protein activity. This chapter selectively details the best-characterized examples of small noncoding regulatory RNAs (sRNAs) and their molecular function in bacterial pathogens of special interest. Salmonella enterica serovar Typhimurium is a gram-negative pathogen causing gastroenteritis in humans. The two most recent additions to the list of Vibrio sRNA regulators affecting pathogenicity are TarA and TarB, both of which are controlled by the master virulence regulator ToxT. Virulence of Pseudomonas aeruginosa is modulated by quorum sensing (QS) systems that control the production of several virulence factors in a cell density-dependent manner. Posttranscriptional control by the RNA-binding protein RsmA (CsrA) regulates many virulence genes of P. aeruginosa. There are a few notable exceptions, including RNAIII in Staphylococcus, IsrM in Salmonella, and several sRNAs in Listeria, deletion of which leads to clear virulence phenotypes. While many functionally related virulence factors are often clustered in horizontally acquired pathogenicity islands, trans-acting sRNAs located in the ancestral genome can be co-opted into regulation of horizontally acquired genes, thus linking expression of virulence factors with regulation of the core genome.

This chapter highlights some of the many interesting cases of negative regulation at work during different stages of infection for several pathogens, focusing on the diverse molecular mechanisms involved in gene repression and the selective pressures that led to their evolution. Some pathogenic bacteria cycle through multiple hosts; for example, some pathogens use arthropod vectors to invade human populations, an extreme transition that demands a great deal of regulatory flexibility. During infection, pathogenic bacteria must contend with an in vivo environment that is under the surveillance of immune mechanisms capable of rapidly identifying and eliminating foreign microorganisms. Immune recognition presents a significant challenge to pathogenic bacteria. A central mechanism to evade host defenses is to stop producing the structures, such as flagella and pili, that are recognized by host antibodies and toll-like receptors (TLRs). During infection of a new host, flagellar breakage allows the derepression of virulence again. Negative regulation in transcriptional programs in vivo has also been found in Salmonella enterica, a gram-negative pathogen that is the cause of disease in humans and other mammalian hosts. Virulence genes are frequently encoded in clusters on the genomes of pathogens, and these clusters are termed “pathogenicity islands". The deactivation of CovR has been shown to be mediated through selection of CovS mutants in the presence of innate immune responses, but it also appears to be regulated by as-yet-unknown signals sensed by CovS.

To survive in their environment, bacteria must sense the conditions in which they live and adapt accordingly. The best way to acquire this information is to sense various molecules produced by the host. This interaction, along with the host sensing bacterium-derived molecules, has been termed interkingdom signaling. Hormones, the principal signaling molecules in multicellular organisms, are present in every environment where bacteria interact with the host. Catecholamines are a family of three neuroendocrine hormones that are produced from the amino acid tyrosine. Opioids are neurotransmitters that play multiple roles in host responses such as stress, tissue damage, and regulation of the immune system. Natriuretic peptides are a class of peptide hormones involved in the osmoregulation of blood. The major signaling molecules used by the immune system are cytokines, which makes them a prime target for being eavesdropped on by bacteria. The immune system produces many other molecules aside from cytokines. Among these are cationic antimicrobial peptides (CAMPs). These peptides have a wide array of diversity but share a positive charge and amphipathic regions that allow them to interact with and disrupt the negatively charged bacterial membrane. Gram-positive bacteria also must face the wide array of antimicrobial peptides produced by the host. The ability to sense and respond to host signals has been identified in most major pathogens. The prevalence of this ability suggests that interkingdom signaling may be extremely common and that many more systems will be uncovered in the future.

Properly mediating the transition between host and environment is of particular importance to toxigenic Vibrio cholerae, as it encounters numerous physical and biological stresses during these transitions. Interestingly, there is evidence that V. cholerae has evolved to preemptively prepare itself for this transition by turning off virulence genes and turning on environmental survival genes while still in the human host. This chapter focuses heavily on the genetic changes the bacterium undergoes as it transitions out of the host and into the aquatic environment. To better understand the changes in V. cholerae gene expression that occur during the transition out of the host, the chapter first discusses the state of gene expression prior to this transition. This prior, acute stage of infection within the small intestine is when V. cholerae multiplies on the epithelium and expresses virulence factors. Over the course of an infection, waterborne pathogens undergo two major transitions, environment to host and host to environment. During both of these transitions, they experience major physiochemical and nutrient stresses. The chapter focuses on the model pathogen V. cholerae, whose life cycle is studied in depth, and reveals fascinating adaptive and evolutionary strategies for moving between host and environment. Determination of where and how V. cholerae and other waterborne pathogens are persisting in the environment and the mechanisms by which they cycle between environment and host will allow us to more rationally plan public health interventions with maximum effectiveness.