Menstrual toxic shock syndrome (mTSS) is caused by strains of Staphylococcus aureus that overproduce a toxin known as toxic shock syndrome toxin-1 (TSST-1). This work studied how glucose levels in a model vaginal environment could influence the amount of TSST-1 that is produced by S. aureus .

Staphylococcus aureus infections can lead to diseases that range from localized skin abscess to life-threatening toxic shock syndrome. The SrrAB two-component system (TCS) is a global regulator of S. aureus virulence and critical for survival under environmental conditions such as hypoxic, oxidative, and nitrosative stress found at sites of infection. Despite the critical role of SrrAB in S. aureus pathogenicity, the mechanism by which the SrrAB TCS senses and responds to these environmental signals remains unknown. Bioinformatics analysis showed that the SrrB histidine kinase contains several domains, including an extracellular Cache domain and a cytoplasmic HAMP-PAS-DHp-CA region. Here, we show that the PAS domain regulates both kinase and phosphatase enzyme activity of SrrB and present the structure of the DHp-CA catalytic core. Importantly, this structure shows a unique intramolecular cysteine disulfide bond in the ATP-binding domain that significantly affects autophosphorylation kinetics. In vitro data show that the redox state of the disulfide bond affects S. aureus biofilm formation and toxic shock syndrome toxin-1 production. Moreover, with the use of the rabbit infective endocarditis model, we demonstrate that the disulfide bond is a critical regulatory element of SrrB function during S. aureus infection. Our data support a model whereby the disulfide bond and PAS domain of SrrB sense and respond to the cellular redox environment to regulate S. aureus survival and pathogenesis.

Toxic shock syndrome toxin-1 (TSST-1) is a superantigen (SAg) produced by Staphylococcus aureus thought to be responsible for essentially all cases of menstrual-associated toxic shock syndrome (TSS). As a potent exotoxin, it is not surprising that S. aureus has evolved multiple systems to control expression of TSST-1. Although the accessory gene regulator (Agr) system is recognized to enhance TSST-1 expression, how Agr regulates TSST-1 is unclear. Using an agr-null mutant, complementation experiments demonstrated that Agr controls TSST-1 expression through the activity of the RNAIII effector molecule. RNAIII can repress translation of the repressor of toxins (Rot) regulator, and deletion of rot increased expression of TSST-1 during the exponential phase of growth. Deletion of agr did not affect rot transcription, but did result in overexpression of the Rot protein, and Rot was also shown to bind and positively regulate the rot promoter. Overexpression of Rot dramatically repressed TSST-1, and Rot bound directly to the TSST-1 promoter. Deletion of both agr and rot in S. aureus returned TSST-1 expression to wild-type levels. This work demonstrates that Agr, although widely considered to be an inducer of TSST-1, has evolved in combination with Rot, to restrict the expression of this potent SAg.

Streptococcus pyogenes is a globally prominent bacterial pathogen that exhibits strict tropism for the human host, yet bacterial factors responsible for the ability of S. pyogenes to compete within this limited biological niche are not well understood. Using an engineered recombinase-based in vivo expression technology (RIVET) system, we identified an in vivo-induced promoter region upstream of a predicted Class IIb bacteriocin system in the M18 serotype S. pyogenes strain MGAS8232. This promoter element was not active under in vitro laboratory conditions, but was highly induced within the mouse nasopharynx. Recombinant expression of the predicted mature S. pyogenes bacteriocin peptides (designated SpbM and SpbN) revealed that both peptides were required for antimicrobial activity. Using a gain of function experiment in Lactococcus lactis, we further demonstrated S. pyogenes immunity function is encoded downstream of spbN. These data highlight the importance of bacterial gene regulation within appropriate environments to help understand mechanisms of niche adaptation by bacterial pathogens.

ABSTRACT Toxic shock syndrome toxin 1 (TSST-1) is a Staphylococcus aureus superantigen that has been implicated in both menstrual and nonmenstrual toxic shock syndrome (TSS). Despite the important role of TSST-1 in severe human disease, a comprehensive understanding of staphylococcal regulatory factors that control TSST-1 expression remains incomplete. The S. aureus exotoxin expression (Sae) operon contains a well-characterized two-component system that regulates a number of important exotoxins in S. aureus , although regulation of TSST-1 by the Sae system has not been investigated. We generated a defined deletion mutant of the Sae histidine kinase sensor ( saeS ) in the prototypic menstrual TSS strain S. aureus MN8. Mutation of saeS resulted in a complete loss of TSST-1 expression. Using both luciferase reporter experiments and quantitative real-time PCR, we demonstrate that the Sae system is an important transcriptional activator of TSST-1 expression. Recombinant SaeR was able to bind directly to the tst promoter to a region containing two SaeR consensus binding sites. Although the stand-alone SarA transcriptional regulator has been shown to be both a positive and a negative regulator of TSST-1, deletion of sarA in S. aureus MN8 resulted in a dramatic overexpression of TSST-1. As expected, mutation of agr also reduced TSST-1 expression, but this phenotype appeared to be independent of Sae. A double mutation of saeS and sarA resulted in the loss of TSST-1 expression. This work indicates that the Sae system is a dominant and direct transcriptional activator that is required for expression of TSST-1. IMPORTANCE The TSST-1 superantigen is an exotoxin, produced by some strains of S. aureus , that has a clear role in both menstrual and nonmenstrual TSS. Although the well-characterized agr quorum sensing system is a known positive regulator of TSST-1, the molecular mechanisms that directly control TSST-1 expression are only partially understood. Our studies demonstrate that the Sae two-component regulatory system is a positive transcriptional regulator that binds directly to the TSST-1 promoter, and furthermore, our data suggest that Sae is required for expression of TSST-1. This work highlights how major regulatory circuits can converge to fine-tune exotoxin expression and suggests that the Sae regulatory system may be an important target for antivirulence strategies.

Superantigens (SAgs) are microbial toxins defined by their ability to activate T lymphocytes in a T cell receptor (TCR) β-chain variable domain (Vβ)-specific manner. Although existing structural information indicates that diverse bacterial SAgs all uniformly engage the Vβ second complementarity determining region (CDR2β) loop, the molecular rules that dictate SAg-mediated T cell activation and Vβ specificity are not fully understood. Herein we report the crystal structure of human Vβ2.1 (hVβ2.1) in complex with the toxic shock syndrome toxin-1 (TSST-1) SAg, and mutagenesis of hVβ2.1 indicates that the non-canonical length of CDR2β is a critical determinant for recognition by TSST-1 as well as the distantly related SAg streptococcal pyrogenic exotoxin C. Frame work (FR) region 3 is uniquely critical for TSST-1 function explaining the fine Vβ-specificity exhibited by this SAg. Furthermore, domain swapping experiments with SAgs, which use distinct domains to engage both CDR2β and FR3/4β revealed that the CDR2β contacts dictate T lymphocyte Vβ-specificity. These findings demonstrate that the TCR CDR2β loop is the critical determinant for functional recognition and Vβ-specificity by diverse bacterial SAgs.

A. K. M. Nur-ur Rahman, Daniel A. Bonsor, Christine A. Herfst , Fraser Pollard, Michael Peirce, Aaron W. Wyatt, Katherine J. Kasper, Joaquín Madrenas**, Eric J. Sundberg, and John K. McCormick 2 From the Department of Microbiology and Immunology and the Centre for Human Immunology, The University of Western Ontario, London, Ontario N6A 5C1, Canada, the Boston Biomedical Research Institute, Watertown, Massachusetts 02472, the Lawson Health Research Institute, St. Joseph’s Health Care, London, Ontario N6A 4V2, Canada, and **Robarts Research Institute, The University of Western Ontario, London, Ontario N6A 5K8, Canada

Superantigens (SAGs) bind simultaneously to major histocompatibility complex (MHC) and T-cell receptor (TCR) molecules, resulting in the massive release of inflammatory cytokines that can lead to toxic shock syndrome (TSS) and death. A major causative agent of TSS is toxic shock syndrome toxin-1 (TSST-1), which is unique relative to other bacterial SAGs owing to its structural divergence and its stringent TCR specificity. Here, we report the crystal structure of TSST-1 in complex with an affinity-matured variant of its wild-type TCR ligand, human T-cell receptor beta chain variable domain 2.1. From this structure and a model of the wild-type complex, we show that TSST-1 engages TCR ligands in a markedly different way than do other SAGs. We provide a structural basis for the high TCR specificity of TSST-1 and present a model of the TSST-1-dependent MHC-SAG-TCR T-cell signaling complex that is structurally and energetically unique relative to those formed by other SAGs. Our data also suggest that protein plasticity plays an exceptionally significant role in this affinity maturation process that results in more than a 3000-fold increase in affinity.

Superantigens (SAgs) are potent microbial toxins that bind simultaneously to T cell receptors (TCRs) and class II major histocompatibility complex molecules, resulting in the activation and expansion of large T cell subsets and the onset of numerous human diseases. Within the bacterial SAg family, streptococcal pyrogenic exotoxin I (SpeI) has been classified as belonging to the group V SAg subclass, which are characterized by a unique, relatively conserved similar to 15 amino acid extension (amino acid residues 154 to 170 in SpeI; herein referred to as the alpha 3-beta 8 loop), absent in SAg groups I through IV. Here, we report the crystal structure of Spel at 1.56 A resolution. Although the alpha 3-beta 8 loop in Spel is several residues shorter than that of another group V SAg, staphylococcal enterotoxin serotype I, the C-terminal portions of these loops, which are located adjacent to the putative TCR binding site, are structurally similar. Mutagenesis and subsequent functional analysis of Spel indicates that TCR beta-chains are likely engaged in a similar general orientation as other characterized SAgs. We show, however, that the alpha 3-beta 8 loop length, and the presence of key glycine residues, are necessary for optimal activation of T cells. Based on V beta-skewing analysis of human T cells activated with Spel and structural models, we propose that the alpha 3-beta 8 loop is positioned to form productive intermolecular contacts with the TCR chain, likely in framework region 3, and that these contacts are required for optimal TCR recognition by Spel, and likely all other group V SAgs. (c) 2007 Elsevier Ltd. All rights reserved.

Although cellular processes depend on protein-protein interactions, our understanding of molecular recognition between proteins remains far from comprehensive. Protein-protein interfaces are structural and energetic mosaics in which a subset of interfacial residues, called hot spots, contributes disproportionately to the affinity of the complex. These hot-spot residues can be further clustered into hot regions. It has been proposed that binding energetics between residues within a hot region are cooperative, whereas those between hot regions are strictly additive. If this idea held true for all protein-protein interactions, then energetically significant long-range conformational effects would be unlikely to occur. In the present study, we show cooperative binding energetics between distinct hot regions that are separated by > 20 angstrom. Using combinatorial mutagenesis and surface plasmon resonance binding analysis to dissect additivity and cooperativity in a complex formed between a variable domain of a T cell receptor and a bacterial superantigen, we find that combinations of mutations from each of two hot regions exhibited significant cooperative energetics. Their connecting sequence is composed primarily of a single P-strand of the T cell receptor variable Ig domain, which has been observed to undergo a strand-switching event and does not form an integral part of the stabilizing core of this Ig domain. We propose that these cooperative effects are propagated through a dynamic structural network. Cooperativity between hot regions has significant implications for the prediction and inhibition of protein-protein interactions.

Superantigens activate large fractions of T cells through unconventional interactions with both TCR beta-chain V domains (V beta s) and MHC class II molecules. The bacterial superantigen streptococcal pyrogenic exotoxin C (SpeC) primarily stimulates human V beta 2(+) T cells. Herein, we have analyzed the SpeC-V beta 2.1 interaction by mutating all SpeC residues that make contact with V beta 2.1 and have determined the energetic and functional consequences of these mutations. Our comprehensive approach, including mutagenesis, functional readouts from both bulk T cell populations, and an engineered V beta 2.1(+) Jurkat T cell, as well as surface plasmon resonance binding analysis, has defined the SpeC "functional epitope" for TCR engagement. Although only two SpeC residues (Tyr(15) and Arg(181)) are critical for activation of virtually all human CD3(+) T cells, a larger cluster of four hot spot residues are required for interaction with V beta 2.1. Three of these residues (Tyr(15), Phe(75) and Arg(181)) concentrate their binding energy on the CDR2 loop residue Ser(52a), a noncanonical residue insertion found only in V beta 2 and V beta 4 chains. Plasticity of this loop is important for recognition by SpeC. Although SpeC interacts with the V beta 2.1 hypervariable CDR3 loop, our data indicate these contacts have little to no influence on the functional interaction with V beta 2.1. These studies also provide a molecular basis for selectivity and cross-reactivity of SpeC-TCR recognition and reveal a degree of fine specificity in these interactions, whereby certain SpeC mutants are capable of distinguishing between different alleles of the same V beta domain subfamily.

Type III secretion systems are utilized by a number of gram-negative bacterial pathogens to deliver virulence-associated proteins into host cells. Using a PCR-based approach, we identified homologs of type III secretion genes in the gram-negative bacterium Burkholderia cepacia, an important pulmonary pathogen in, immunocompromised patients and patients with cystic fibrosis. One of the genes, designated bscN, encodes a member of a family of ATP-binding proteins believed to generate energy driving virulence protein secretion. Genetic dissection of the regions flanking the bscN gene revealed a locus consisting of at least 10 open reading frames, predicted to encode products with significant homology to known type III secretion proteins in other bacteria. A defined null mutation was generated in the bscN gene, and the null strain and wild-type parent strain were examined by use of a murine model of B. cepacia infection. Quantitative bacteriological analysis of the lungs and spleens of infected C57BL/6 mice revealed that the bscN null strain was attenuated in virulence compared to the parent strain, with significantly lower bacterial recovery from the lungs and spleens at 3 days postinfection. Moreover, histopathological changes, including an inflammatory cell infiltrate, were more pronounced in the lungs of mice infected with the wild-type parent strain than in those of mice infected with the isogenic bscN mutant. These results implicate type III secretion as an important determinant in the pathogenesis of B. cepacia.

ABSTRACT Burkholderia cepacia is an important opportunistic human pathogen that affects immunocompromised individuals, particularly cystic fibrosis (CF) patients. Colonization of the lungs of a CF patient by B. cepacia can lead not only to a decline in respiratory function but also to an acute systemic infection, such as bacteremia. We have previously demonstrated that a CF clinical isolate of B. cepacia, strain J2315, can invade and survive within cultured respiratory epithelial cells. In order to further characterize the mechanisms of invasion of B. cepacia, we screened a transposon-generated mutant library of strain J2315 for mutants defective in invasion of A549 respiratory epithelial cells. Here we describe isolation and characterization of a nonmotile mutant of B. cepacia with reduced invasiveness due to disruption of fliG, which encodes a component of the motor-switch complex of the flagellar basal body. We also found that a defined null mutation in fliI, a gene encoding a highly conserved ATPase required for protein translocation via the flagellar type III secretion system, also resulted in loss of motility and a significant reduction in invasion. Both mutants lacked detectable intracellular flagellin and failed to export detectable amounts of flagellin into culture supernatants, suggesting that disruption of fliG and fliI impaired flagellar biogenesis. The reduction in invasion did not appear to be due to defective adherence of the flagellar mutants to A549 cells, suggesting that functional flagella and motility are required for full invasiveness of B. cepacia. Our findings indicate that flagellum-mediated motility may facilitate penetration of host epithelial barriers by B. cepacia, contributing to establishment of infection and systemic spread of the organism.

The Gram-negative bacterium Burkholderia cepacia has recently emerged as an important opportunistic pathogen in humans. This review focuses on the cellular aspects of B. cepacia infection and the dynamics of the B. cepacia-host cell interaction, including recent advances in our understanding of the ability of B. cepacia to adhere to, enter, and survive intracellularly within human cells.

The Gram-negative bacterium Burkholderia cepacia has recently emerged as an important opportunistic pathogen in humans. This review focuses on the cellular aspects of B. cepacia infection and the dynamics of the B. cepacia–host cell interaction, including recent advances in our understanding of the ability of B. cepacia to adhere to, enter, and survive intracellularly within human cells.