In Situ Molecular Architecture of the Salmonella Type III Secretion Machine.

Type III protein secretion systems have specifically evolved to deliver bacterially encoded proteins into target eukaryotic cells. The core elements of this multi-protein machine are the envelope-associated needle complex, the inner membrane export apparatus, and a large cytoplasmic sorting platform. Here, we report a high-resolution in situ structure of the Salmonella Typhimurium type III secretion machine obtained by high-throughput cryo-electron tomography and sub-tomogram averaging. Through molecular modeling and comparative analysis of machines assembled with protein-tagged components or from different deletion mutants, we determined the molecular architecture of the secretion machine in situ and localized its structural components. We also show that docking of the sorting platform results in significant conformational changes in the needle complex to provide the symmetry adaptation required for the assembly of the entire secretion machine. These studies provide major insight into the structure and assembly of a broadly distributed protein secretion machine.

Structural Biology of Bacterial Type IV Secretion Systems.

Type IV secretion systems (T4SSs) are large multisubunit translocons, found in both gram-negative and gram-positive bacteria and in some archaea. These systems transport a diverse array of substrates from DNA and protein-DNA complexes to proteins, and play fundamental roles in both bacterial pathogenesis and bacterial adaptation to the cellular milieu in which bacteria live. This review describes the various biochemical and structural advances made toward understanding the biogenesis, architecture, and function of T4SSs.

Ubiquitin-targeted bacterial effectors: rule breakers of the ubiquitin system.

Regulation through post-translational ubiquitin signaling underlies a large portion of eukaryotic biology. This has not gone unnoticed by invading pathogens, many of which have evolved mechanisms to manipulate or subvert the host ubiquitin system. Bacteria are particularly adept at this and rely heavily upon ubiquitin-targeted virulence factors for invasion and replication. Despite lacking a conventional ubiquitin system of their own, many bacterial ubiquitin regulators loosely follow the structural and mechanistic rules established by eukaryotic ubiquitin machinery. Others completely break these rules and have evolved novel structural folds, exhibit distinct mechanisms of regulation, or catalyze foreign ubiquitin modifications. Studying these interactions can not only reveal important aspects of bacterial pathogenesis but also shed light on unexplored areas of ubiquitin signaling and regulation. In this review, we discuss the methods by which bacteria manipulate host ubiquitin and highlight aspects that follow or break the rules of ubiquitination.

Catalytically Active Cas9 Mediates Transcriptional Interference to Facilitate Bacterial Virulence.

In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely represents a broad paradigm of regulatory functionality, which is potentially critical to the physiology of numerous Cas9-encoding pathogenic and commensal organisms.

Conformational ensembles in Klebsiella pneumoniae FimH impact uropathogenesis.

Klebsiella pneumoniae is an important pathogen causing difficult-to-treat urinary tract infections (UTIs). Over 1.5 million women per year suffer from recurrent UTI, reducing quality of life and causing substantial morbidity and mortality, especially in the hospital setting. Uropathogenic E. coli (UPEC) is the most prevalent cause of UTI. Like UPEC, K. pneumoniae relies on type 1 pili, tipped with the mannose-binding adhesin FimH, to cause cystitis. However, K. pneumoniae FimH is a poor binder of mannose, despite a mannose-binding pocket identical to UPEC FimH. FimH is composed of two domains that are in an equilibrium between tense (low-affinity) and relaxed (high-affinity) conformations. Substantial interdomain interactions in the tense conformation yield a low-affinity, deformed mannose-binding pocket, while domain-domain interactions are broken in the relaxed state, resulting in a high-affinity binding pocket. Using crystallography, we identified the structural basis by which domain-domain interactions direct the conformational equilibrium of K. pneumoniae FimH, which is strongly shifted toward the low-affinity tense state. Removal of the pilin domain restores mannose binding to the lectin domain, thus showing that poor mannose binding by K. pneumoniae FimH is not an inherent feature of the mannose-binding pocket. Phylogenetic analyses of K. pneumoniae genomes found that FimH sequences are highly conserved. However, we surveyed a collection of K. pneumoniae isolates from patients with long-term indwelling catheters and identified isolates that possessed relaxed higher-binding FimH variants, which increased K. pneumoniae fitness in bladder infection models, suggesting that long-term residence within the urinary tract may select for higher-binding FimH variants.

An interstrand DNA crosslink glycosylase aids Acinetobacter baumannii pathogenesis.

Maintenance of DNA integrity is essential to all forms of life. DNA damage generated by reaction with genotoxic chemicals results in deleterious mutations, genome instability, and cell death. Pathogenic bacteria encounter several genotoxic agents during infection. In keeping with this, the loss of DNA repair networks results in virulence attenuation in several bacterial species. Interstrand DNA crosslinks (ICLs) are a type of DNA lesion formed by covalent linkage of opposing DNA strands and are particularly toxic as they interfere with replication and transcription. Bacteria have evolved specialized DNA glycosylases that unhook ICLs, thereby initiating their repair. In this study, we describe AlkX, a DNA glycosylase encoded by the multidrug resistant pathogen Acinetobacter baumannii. AlkX exhibits ICL unhooking activity similar to that of its Escherichia coli homolog YcaQ. Interrogation of the in vivo role of AlkX revealed that its loss sensitizes cells to DNA crosslinking and impairs A. baumannii colonization of the lungs and dissemination to distal tissues during pneumonia. These results suggest that AlkX participates in A. baumannii pathogenesis and protects the bacterium from stress conditions encountered in vivo. Consistent with this, we found that acidic pH, an environment encountered during host colonization, results in A. baumannii DNA damage and that alkX is induced by, and contributes to, defense against acidic conditions. Collectively, these studies reveal functions for a recently described class of proteins encoded in a broad range of pathogenic bacterial species.

Mechanism of secretion of TcpF by the Vibrio cholerae toxin-coregulated pilus.

Many bacteria possess dynamic filaments called Type IV pili (T4P) that perform diverse functions in colonization and dissemination, including host cell adhesion, DNA uptake, and secretion of protein substrates-exoproteins-from the periplasm to the extracellular space. The Vibrio cholerae toxin-coregulated pilus (TCP) and the enterotoxigenic Escherichia coli CFA/III pilus each mediates export of a single exoprotein, TcpF and CofJ, respectively. Here, we show that the disordered N-terminal segment of mature TcpF is the export signal (ES) recognized by TCP. Deletion of the ES disrupts secretion and causes TcpF to accumulate in the V. cholerae periplasm. The ES alone can mediate export of Neisseria gonorrhoeae FbpA by V. cholerae in a T4P-dependent manner. The ES is specific for its autologous T4P machinery as CofJ bearing the TcpF ES is exported by V. cholerae, whereas TcpF bearing the CofJ ES is not. Specificity is mediated by binding of the ES to TcpB, a minor pilin that primes pilus assembly and forms a trimer at the pilus tip. Finally, the ES is proteolyzed from the mature TcpF protein upon secretion. Together, these results provide a mechanism for delivery of TcpF across the outer membrane and release into the extracellular space.

High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo.

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

A novel phospholipase A2 is a core component of the typhoid toxin genetic islet.

S. Typhi, the cause of typhoid fever, is a bacterial pathogen of substantial global importance. Typhoid toxin is a secreted AB-type toxin that is a key S. Typhi virulence factor encoded within a 5-gene genetic islet. Four genes in this islet have well-defined roles in typhoid toxin biology, however the function of the fifth gene is unknown. Here, we investigate the function of this gene, which we name ttaP. We show that ttaP is co-transcribed with the typhoid toxin subunit cdtB, and we perform genomic analyses that indicate that TtaP is very highly conserved in typhoid toxin islets found in diverse salmonellae. We show that TtaP is a distant homolog of group XIV secreted phospholipase A2 (PLA2) enzymes, and experimentally demonstrate that TtaP is a bona fide PLA2. Sequence and structural analyses indicate that TtaP differs substantially from characterized PLA2s, and thus represents a novel class of PLA2. Secretion assays revealed that TtaP is neither co-secreted with typhoid toxin, nor is it required for toxin secretion. Although TtaP is a phospholipase that remains associated with the S. Typhi cell, assays that probed for altered cell envelope integrity failed to identify any differences between wild-type S. Typhi and a ttaP deletion strain. Collectively, this study identifies a biochemical activity for the lone uncharacterized typhoid toxin islet gene and lays the groundwork for exploring how this gene factors into S. Typhi pathogenesis. This study further identifies a novel class of PLA2, enzymes that have a wide range of industrial applications.

Structural and functional characterization of the IpaD π-helix reveals critical roles in DOC interaction, T3SS apparatus maturation, and Shigella virulence.

Shigella spp. are highly pathogenic members of the Enterobacteriaceae family, causing ∼269 million cases of bacillary dysentery and >200,000 deaths each year. Like many Gram-negative pathogens, Shigella rely on their type three secretion system (T3SS) to inject effector proteins into eukaryotic host cells, driving both cellular invasion and evasion of host immune responses. Exposure to the bile salt deoxycholate (DOC) significantly enhances Shigella virulence and is proposed to serve as a critical environmental signal present in the small intestine that prepares Shigella's T3SS for efficient infection of the colonic epithelium. Here, we uncover critical mechanistic details of the Shigella-specific DOC signaling process by describing the role of a π-helix secondary structure element within the T3SS tip protein invasion plasmid antigen D (IpaD). Biophysical characterization and high-resolution structures of IpaD mutants lacking the π-helix show that it is not required for global protein structure, but that it defines the native DOC binding site and prevents off target interactions. Additionally, Shigella strains expressing the π-helix deletion mutants illustrate the pathogenic importance of its role in guiding DOC interaction as flow cytometry and gentamycin protection assays show that the IpaD π-helix is essential for DOC-mediated apparatus maturation and enhanced invasion of eukaryotic cells. Together, these findings add to our understanding of the complex Shigella pathogenesis pathway and its evolution to respond to environmental bile salts by identifying the π-helix in IpaD as a critical structural element required for translating DOC exposure to virulence enhancement.

The extracellular vesicle generation paradox: a bacterial point of view.

All bacteria produce secreted vesicles that carry out a variety of important biological functions. These extracellular vesicles can improve adaptation and survival by relieving bacterial stress and eliminating toxic compounds, as well as by facilitating membrane remodeling and ameliorating inhospitable environments. However, vesicle production comes with a price. It is energetically costly and, in the case of colonizing pathogens, it elicits host immune responses, which reduce bacterial viability. This raises an interesting paradox regarding why bacteria produce vesicles and begs the question as to whether the benefits of producing vesicles outweigh their costs. In this review, we discuss the various advantages and disadvantages associated with Gram-negative and Gram-positive bacterial vesicle production and offer perspective on the ultimate score. We also highlight questions needed to advance the field in determining the role for vesicles in bacterial survival, interkingdom communication, and virulence.

The sorting platform in the type III secretion pathway: From assembly to function.

The type III secretion system (T3SS) is a specialized nanomachine that enables bacteria to secrete proteins in a specific order and directly deliver a specific set of them, collectively known as effectors, into eukaryotic organisms. The core structure of the T3SS is a syringe-like apparatus composed of multiple building blocks, including both membrane-associated and soluble proteins. The cytosolic components organize together in a chamber-like structure known as the sorting platform (SP), responsible for recruiting, sorting, and initiating the substrates destined to engage this secretion pathway. In this article, we provide an overview of recent findings on the SP's structure and function, with a particular focus on its assembly pathway. Furthermore, we discuss the molecular mechanisms behind the recruitment and hierarchical sorting of substrates by this cytosolic complex. Overall, the T3SS is a highly specialized and complex system that requires precise coordination to function properly. A deeper understanding of how the SP orchestrates T3S could enhance our comprehension of this complex nanomachine, which is central to the host-pathogen interface, and could aid in the development of novel strategies to fight bacterial infections.

Phenol-soluble modulins PSMα3 and PSMß2 form nanotubes that are cross-α amyloids.

Phenol-soluble modulins (PSMs) are peptide-based virulence factors that play significant roles in the pathogenesis of staphylococcal strains in community-associated and hospital-associated infections. In addition to cytotoxicity, PSMs display the propensity to self-assemble into fibrillar species, which may be mediated through the formation of amphipathic conformations. Here, we analyze the self-assembly behavior of two PSMs, PSMα3 and PSMß2, which are derived from peptides expressed by methicillin-resistant Staphylococcus aureus (MRSA), a significant human pathogen. In both cases, we observed the formation of a mixture of self-assembled species including twisted filaments, helical ribbons, and nanotubes, which can reversibly interconvert in vitro. Cryo­electron microscopy structural analysis of three PSM nanotubes, two derived from PSMα3 and one from PSMß2, revealed that the assemblies displayed remarkably similar structures based on lateral association of cross-α amyloid protofilaments. The amphipathic helical conformations of PSMα3 and PSMß2 enforced a bilayer arrangement within the protofilaments that defined the structures of the respective PSMα3 and PSMß2 nanotubes. We demonstrate that, similar to amyloids based on cross-ß protofilaments, cross-α amyloids derived from these PSMs display polymorphism, not only in terms of the global morphology (e.g., twisted filament, helical ribbon, and nanotube) but also with respect to the number of protofilaments within a given peptide assembly. These results suggest that the folding landscape of PSM derivatives may be more complex than originally anticipated and that the assemblies are able to sample a wide range of supramolecular structural space.

Reconstitution of the S. aureus agr quorum sensing pathway reveals a direct role for the integral membrane protease MroQ in pheromone biosynthesis.

In Staphylococcus aureus, virulence is under the control of a quorum sensing (QS) circuit encoded in the accessory gene regulator (agr) genomic locus. Key to this pathogenic behavior is the production and signaling activity of a secreted pheromone, the autoinducing peptide (AIP), generated following the ribosomal synthesis and posttranslational modification of a precursor polypeptide, AgrD, through two discrete cleavage steps. The integral membrane protease AgrB is known to catalyze the first processing event, generating the AIP biosynthetic intermediate, AgrD (1-32) thiolactone. However, the identity of the second protease in this biosynthetic pathway, which removes an N-terminal leader sequence, has remained ambiguous. Here, we show that membrane protease regulator of agr QS (MroQ), an integral membrane protease recently implicated in the agr response, is directly involved in AIP production. Genetic complementation and biochemical experiments reveal that MroQ proteolytic activity is required for AIP biosynthesis in agr specificity group I and group II, but not group III. Notably, as part of this effort, the biosynthesis and AIP-sensing arms of the QS circuit were reconstituted together in vitro. Our experiments also reveal the molecular features guiding MroQ cleavage activity, a critical factor in defining agr specificity group identity. Collectively, our study adds to the molecular understanding of the agr response and Staphylococcus aureus virulence.

TolC and EmrA1 contribute to Francisella novicida multidrug resistance and modulation of host cell death.

Francisella spp. are Gram-negative, facultative intracellular pathogens. Francisella tularensis causes the human disease tularemia and is considered a biological threat agent due to its high infectivity and virulence. A central aspect of Francisella virulence is its ability to dampen host immune responses. We previously identified the outer membrane channel (OMC) protein TolC as a critical F. tularensis virulence factor required for suppression of apoptotic and proinflammatory responses during macrophage infection. TolC functions as part of multidrug efflux systems and the type I secretion pathway that exports bacterial effector proteins. In these systems, TolC forms tripartite complexes together with an inner membrane transporter and periplasmic membrane fusion protein (MFP). To advance understanding of TolC function in Francisella, we analyzed OMC and MFP homologs in Francisella novicida, a widely used model species that causes a tularemia-like disease in mice. In agreement with the previous F. tularensis studies, all three OMCs present in F. novicida contributed to multidrug resistance, but only TolC was important for suppressing macrophage cell death. In addition, we identified the EmrA1 MFP as important for resisting antimicrobial compounds and dampening host cell death. In contrast to results obtained with F. tularensis, the cell death triggered during infection with the F. novicida tolC and emrA1 mutants was dominated by pyroptosis rather than apoptosis. These data expand our understanding of TolC function in Francisella and underscore both conserved and differential aspects of F. novicida and F. tularensis. IMPORTANCE: Francisella tularensis is a Gram-negative intracellular bacterial pathogen and causative agent of tularemia. We previously identified the outer membrane channel protein TolC as contributing to antimicrobial resistance and subversion of host responses by F. tularensis. To advance understanding of TolC function in Francisella and to identify components that might work together with TolC, we took advantage of a transposon mutant library in F. novicida, a model species that causes a tularemia-like disease in mice. Our findings identify TolC and the membrane fusion protein EmrA1 as important for both antimicrobial resistance and suppression of macrophage cell death. This study also revealed differences in cell death pathways triggered by F. novicida versus F. tularensis infection that may relate to differences in virulence.

The structure of NAD+ consuming protein Acinetobacter baumannii TIR domain shows unique kinetics and conformations.

Toll-like and interleukin-1/18 receptor/resistance (TIR) domain-containing proteins function as important signaling and immune regulatory molecules. TIR domain-containing proteins identified in eukaryotic and prokaryotic species also exhibit NAD+ hydrolase activity in select bacteria, plants, and mammalian cells. We report the crystal structure of the Acinetobacter baumannii TIR domain protein (AbTir-TIR) with confirmed NAD+ hydrolysis and map the conformational effects of its interaction with NAD+ using hydrogen-deuterium exchange-mass spectrometry. NAD+ results in mild decreases in deuterium uptake at the dimeric interface. In addition, AbTir-TIR exhibits EX1 kinetics indicative of large cooperative conformational changes, which are slowed down upon substrate binding. Additionally, we have developed label-free imaging using the minimally invasive spectroscopic method 2-photon excitation with fluorescence lifetime imaging, which shows differences in bacteria expressing native and mutant NAD+ hydrolase-inactivated AbTir-TIRE208A protein. Our observations are consistent with substrate-induced conformational changes reported in other TIR model systems with NAD+ hydrolase activity. These studies provide further insight into bacterial TIR protein mechanisms and their varying roles in biology.

Molecular determinants for differential activation of the bile acid receptor from the pathogen Vibrio parahaemolyticus.

Bile acids are important for digestion of food and antimicrobial activity. Pathogenic Vibrio parahaemolyticus senses bile acids and induce pathogenesis. The bile acid taurodeoxycholate (TDC) was shown to activate the master regulator, VtrB, of this system, whereas other bile acids such as chenodeoxycholate (CDC) do not. Previously, VtrA-VtrC was discovered to be the co-component signal transduction system that binds bile acids and induces pathogenesis. TDC binds to the periplasmic domain of the VtrA-VtrC complex, activating a DNA-binding domain in VtrA that then activates VtrB. Here, we find that CDC and TDC compete for binding to the VtrA-VtrC periplasmic heterodimer. Our crystal structure of the VtrA-VtrC heterodimer bound to CDC revealed CDC binds in the same hydrophobic pocket as TDC but differently. Using isothermal titration calorimetry, we observed that most mutants in the binding pocket of VtrA-VtrC caused a decrease in bile acid binding affinity. Notably, two mutants in VtrC bound bile acids with a similar affinity as the WT protein but were attenuated for TDC-induced type III secretion system 2 activation. Collectively, these studies provide a molecular explanation for the selective pathogenic signaling by V. parahaemolyticus and reveal insight into a host's susceptibility to disease.

Increased intracellular persulfide levels attenuate HlyU-mediated hemolysin transcriptional activation in Vibrio cholerae.

The vertebrate host's immune system and resident commensal bacteria deploy a range of highly reactive small molecules that provide a barrier against infections by microbial pathogens. Gut pathogens, such as Vibrio cholerae, sense and respond to these stressors by modulating the expression of exotoxins that are crucial for colonization. Here, we employ mass spectrometry-based profiling, metabolomics, expression assays, and biophysical approaches to show that transcriptional activation of the hemolysin gene hlyA in V. cholerae is regulated by intracellular forms of sulfur with sulfur-sulfur bonds, termed reactive sulfur species (RSS). We first present a comprehensive sequence similarity network analysis of the arsenic repressor superfamily of transcriptional regulators, where RSS and hydrogen peroxide sensors segregate into distinct clusters of sequences. We show that HlyU, transcriptional activator of hlyA in V. cholerae, belongs to the RSS-sensing cluster and readily reacts with organic persulfides, showing no reactivity or DNA dissociation following treatment with glutathione disulfide or hydrogen peroxide. Surprisingly, in V. cholerae cell cultures, both sulfide and peroxide treatment downregulate HlyU-dependent transcriptional activation of hlyA. However, RSS metabolite profiling shows that both sulfide and peroxide treatment raise the endogenous inorganic sulfide and disulfide levels to a similar extent, accounting for this crosstalk, and confirming that V. cholerae attenuates HlyU-mediated activation of hlyA in a specific response to intracellular RSS. These findings provide new evidence that gut pathogens may harness RSS-sensing as an evolutionary adaptation that allows them to overcome the gut inflammatory response by modulating the expression of exotoxins.

Single-molecule dynamics of DNA gyrase in evolutionarily distant bacteria Mycobacterium tuberculosis and Escherichia coli.

DNA gyrase is an essential nucleoprotein motor present in all bacteria and is a major target for antibiotic treatment of Mycobacterium tuberculosis (MTB) infection. Gyrase hydrolyzes ATP to add negative supercoils to DNA using a strand passage mechanism that has been investigated using biophysical and biochemical approaches. To analyze the dynamics of substeps leading to strand passage, single-molecule rotor bead tracking (RBT) has been used previously to follow real-time supercoiling and conformational transitions in Escherichia coli (EC) gyrase. However, RBT has not yet been applied to gyrase from other pathogenically relevant bacteria, and it is not known whether substeps are conserved across evolutionarily distant species. Here, we compare gyrase supercoiling dynamics between two evolutionarily distant bacterial species, MTB and EC. We used RBT to measure supercoiling rates, processivities, and the geometries and transition kinetics of conformational states of purified gyrase proteins in complex with DNA. Our results show that E. coli and MTB gyrases are both processive, with the MTB enzyme displaying velocities ∼5.5× slower than the EC enzyme. Compared with EC gyrase, MTB gyrase also more readily populates an intermediate state with DNA chirally wrapped around the enzyme, in both the presence and absence of ATP. Our substep measurements reveal common features in conformational states of EC and MTB gyrases interacting with DNA but also suggest differences in populations and transition rates that may reflect distinct cellular needs between these two species.

Revisiting old friends: updates on the role of two-component signaling systems in Listeria monocytogenes survival and pathogenesis.

Listeria monocytogenes is well recognized for both its broad resistance to stress conditions and its ability to transition from a soil bacterium to an intracellular pathogen of mammalian hosts. The bacterium's impressive ability to adapt to changing environments and conditions requires the rapid sensing of environmental cues and the coordinated response of gene products that enable bacterial growth and survival. Two-component signaling systems (TCSs) have been long recognized for their ability to detect environmental stimuli and transmit those signals into transcriptional responses; however, often the precise nature of the stimulus triggering TCS responses can be challenging to define. L. monocytogenes has up to 16 TCSs that have been recognized based on homology and included in this list are several whose functions remain poorly described. This review highlights the current understanding of the breadth and scope of L. monocytogenes TCS as relates to stress resistance and pathogenesis. Precise signals still often remain elusive, but the gene networks associated with TCSs are providing clues into possible functions.