Bacillus anthracis produces a binary toxin composed of protective antigen (PA) and one of two subunits, lethal factor (LF) or edema factor (EF). Most studies have concentrated on induction of toxin-specific antibodies as the correlate of protective immunity, in contrast to which understanding of cellular immunity to these toxins and its impact on infection is limited. We characterized CD4+ T cell immunity to LF in a panel of humanized HLA-DR and DQ transgenic mice and in naturally exposed patients. As the variation in antigen presentation governed by HLA polymorphism has a major impact on protective immunity to specific epitopes, we examined relative binding affinities of LF peptides to purified HLA class II molecules, identifying those regions likely to be of broad applicability to human immune studies through their ability to bind multiple alleles. Transgenics differing only in their expression of human HLA class II alleles showed a marked hierarchy of immunity to LF. Immunogenicity in HLA transgenics was primarily restricted to epitopes from domains II and IV of LF and promiscuous, dominant epitopes, common to all HLA types, were identified in domain II. The relevance of this model was further demonstrated by the fact that a number of the immunodominant epitopes identified in mice were recognized by T cells from humans previously infected with cutaneous anthrax and from vaccinated individuals. The ability of the identified epitopes to confer protective immunity was demonstrated by lethal anthrax challenge of HLA transgenic mice immunized with a peptide subunit vaccine comprising the immunodominant epitopes that we identified.
Group A Streptococcus (GAS) infections represent a major public health burden in both developing and developed countries. In Australia and New Zealand GAS associated diseases are serious problems in Indigenous populations and a major cause of health inequality. Political recognition of these inequalities is providing impetus for strategies that reduce GAS disease and the development of a GAS vaccine now has governmental support in both Australia and New Zealand. Accordingly, an expert workshop was convened in March 2013 to consider available data on GAS vaccines. M-protein based vaccines constructed from the hyper-variable N-terminal region (30-valent vaccine) or the conserved C-repeat domain (J8 vaccine) were reviewed together with vaccine candidates identified using multi high-throughput approaches. Performing a comprehensive assessment of regional GAS strain epidemiology, defining the immune correlates of protection, and the establishment of clinical trial sites were identified as critical activities for a Trans-Tasman vaccine development programme.
Group A streptococcal isolates of serotype M18 are historically associated with epidemic waves of pharyngitis and the non-suppurative immune sequela rheumatic fever. The serotype is defined by a unique, highly encapsulated phenotype, yet the molecular basis for this unusual colony morphology is unknown. Here we identify a truncation in the regulatory protein RocA, unique to and conserved within our serotype M18 GAS collection, and demonstrate that it underlies the characteristic M18 capsule phenotype. Reciprocal allelic exchange mutagenesis of rocA between M18 GAS and M89 GAS demonstrated that truncation of RocA was both necessary and sufficient for hyper-encapsulation via up-regulation of both precursors required for hyaluronic acid synthesis. Although RocA was shown to positively enhance covR transcription, quantitative proteomics revealed RocA to be a metabolic regulator with activity beyond the CovR/S regulon. M18 GAS demonstrated a uniquely protuberant chain formation following culture on agar that was dependent on excess capsule and the RocA mutation. Correction of the M18 rocA mutation reduced GAS survival in human blood, and in vivo naso-pharyngeal carriage longevity in a murine model, with an associated drop in bacterial airborne transmission during infection. In summary, a naturally occurring truncation in a regulator explains the encapsulation phenotype, carriage longevity and transmissibility of M18 GAS, highlighting the close interrelation of metabolism, capsule and virulence.
Streptococcus pyogenes (group A streptococcus, GAS) is responsible for a wide range of pathologies ranging from mild pharyngitis and impetigo to severe invasive soft tissue infections. Despite the continuing susceptibility of the bacterium to ?-lactam antibiotics there has been an unexplained resurgence in the prevalence of invasive GAS infection over the past 30 years. Of particular importance was the emergence of a GAS-associated sepsis syndrome that is analogous to the systemic toxicosis associated with TSST-1 producing strains of Staphylococcus aureus. Despite being recognized for over 20 years, the etiology of GAS associated sepsis and the streptococcal toxic shock syndrome remains poorly understood. Here we review the virulence factors that contribute to the etiology of GAS associated sepsis with a particular focus on coagulation system interactions and the role of the superantigens in the development of streptococcal toxic shock syndrome.
Sepsis is now the leading direct cause of maternal death in the United Kingdom, and Streptococcus pyogenes is the leading pathogen. We combined conventional and genomic analyses to define the duration and scale of a lethal outbreak. Two postpartum deaths caused by S. pyogenes occurred within 24 h; one was characterized by bacteremia and shock and the other by hemorrhagic pneumonia. The women gave birth within minutes of each other in the same maternity unit 2 days earlier. Seven additional infections in health care and household contacts were subsequently detected and treated. All cluster-associated S. pyogenes isolates were genotype emm1 and were initially indistinguishable from other United Kingdom emm1 isolates. Sequencing of the virulence gene sic revealed that all outbreak isolates had the same unique sic type. Genome sequencing confirmed that the cluster was caused by a unique S. pyogenes clone. Transmission between patients occurred on a single day and was associated with casual contact only. A single isolate from one patient demonstrated a sequence change in sic consistent with longer infection duration. Transmission to health care workers was traced to single clinical contacts with index cases. The last case was detected 18 days after the first case. Following enhanced surveillance, the outbreak isolate was not detected again. Mutations in bacterial regulatory genes played no detectable role in this outbreak, illustrating the intrinsic ability of emm1 S. pyogenes to spread while retaining virulence. This fast-moving outbreak highlights the potential of S. pyogenes to cause a range of diseases in the puerperium with rapid transmission, underlining the importance of immediate recognition and response by clinical infection and occupational health teams.
Streptococcus pyogenes infection of the nasopharynx represents a key step in the pathogenic cycle of this organism and a major focus for vaccine development, requiring robust models to facilitate the screening of potentially protective antigens. One antigen that may be an important target for vaccination is the chemokine protease, SpyCEP, which is cell surface-associated and plays a role in pathogenesis. Biophotonic imaging (BPI) can non-invasively characterize the spatial location and abundance of bioluminescent bacteria in vivo. We have developed a bioluminescent derivative of a pharyngeal S. pyogenes strain by transformation of an emm75 clinical isolate with the luxABCDE operon. Evaluation of isogenic recombinant strains in vitro and in vivo confirmed that bioluminescence conferred a growth deficit that manifests as a fitness cost during infection. Notwithstanding this, bioluminescence expression permitted non-invasive longitudinal quantitation of S. pyogenes within the murine nasopharynx albeit with a detection limit corresponding to approximately 10(5) bacterial colony forming units (CFU) in this region. Vaccination of mice with heat killed streptococci, or with SpyCEP led to a specific IgG response in the serum. BPI demonstrated that both vaccine candidates reduced S. pyogenes bioluminescence emission over the course of nasopharyngeal infection. The work suggests the potential for BPI to be used in the non-invasive longitudinal evaluation of potential S. pyogenes vaccines.
Streptococcus pyogenes is a leading cause of pharyngeal infection, with an estimated 616 million cases per year. The human nasopharynx represents the major reservoir for all S. pyogenes infection, including severe invasive disease. To investigate bacterial and host factors that influence S. pyogenes infection, we have devised an improved murine model of nasopharyngeal colonization, with an optimized dosing volume to avoid fulminant infections and a sensitive host strain. In addition we have utilized a refined technique for longitudinal monitoring of bacterial burden that is non-invasive thereby reducing the numbers of animals required. The model was used to demonstrate that the two component regulatory system, CovR/S, is required for optimum infection and transmission from the nasopharynx. There is a fitness cost conferred by covR/S mutation that is specific to the nasopharynx. This may explain why S. pyogenes with altered covR/S have not become prevalent in community infections despite possessing a selective advantage in invasive infection.
Hospital outbreaks of group A streptococcal (GAS) infection can be devastating and occasionally result in the death of previously well patients. Approximately one in ten cases of severe GAS infection is healthcare-associated. This guidance, produced by a multidisciplinary working group, provides an evidence-based systematic approach to the investigation of single cases or outbreaks of healthcare-associated GAS infection in acute care or maternity settings. The guideline recommends that all cases of GAS infection potentially acquired in hospital or through contact with healthcare or maternity services should be investigated. Healthcare workers, the environment, and other patients are possible sources of transmission. Screening of epidemiologically linked healthcare workers should be considered for healthcare-associated cases of GAS infection where no alternative source is readily identified. Communal facilities, such as baths, bidets and showers, should be cleaned and decontaminated between all patients especially on delivery suites, post-natal wards and other high risk areas. Continuous surveillance is required to identify outbreaks which arise over long periods of time. GAS isolates from in-patients, peri-partum patients, neonates, and post-operative wounds should be saved for six months to facilitate outbreak investigation. These guidelines do not cover diagnosis and treatment of GAS infection which should be discussed with an infection specialist.
Sepsis is characterised by a hyper-inflammatory response due to microbial infection. We here review our current understanding of host mechanisms employed to mediate this hyper-inflammatory response, drawing together current knowledge pertaining to pathogen recognition and host pro-inflammatory response. Recognition of microbial derived ligands by pattern recognition receptors (PRRs) is a key step in initiating pro-inflammatory signalling pathways. Examples of PRRs linked to the aetiology of sepsis include Toll-like, C-type lectin, RIG-1-like and also Nod-like receptors, which are involved in the formation of the inflammasome, crucial for the maturation of some pro-inflammatory cytokines. Bacterial superantigens have evolved to exploit host MHC class II and T cell receptors (normally considered part of the adaptive immune response) as innate PRRs to propagate a so-called cytokine storm, while synergy between different microbial ligands and host-derived alarmins can augment the inflammatory response still further through as yet poorly understood interactions. The host pro-inflammatory response results in the characteristic features of inflammation: rubor, calor, dolor, and tumor. We will review herein the key mediators of inflammation in sepsis, identifying their overlapping and intersecting roles in vascular changes in tone, endothelial permeability, coagulation and contact activation, leukocyte mobilisation and activation.
A resurgence of invasive group A streptococcal infections highlights the need for better knowledge of streptococcal biology. This review summarizes the recent advances in our understanding of the field.
Yersinia pestis is the causative agent of plague, a rapidly fatal infectious disease that has not been eradicated worldwide. The capsular Caf1 protein of Y. pestis is a protective antigen under development as a recombinant vaccine. However, little is known about the specificity of human T-cell responses for Caf1. We characterized CD4 T-cell epitopes of Caf1 in "humanized" HLA-DR1 transgenic mice lacking endogenous major histocompatibility complex class II molecules. Mice were immunized with Caf1 or each of a complete set of overlapping synthetic peptides, and CD4 T-cell immunity was measured with respect to proliferative and gamma interferon T-cell responses and recognition by a panel of T-cell hybridomas, as well as direct determination of binding affinities of Caf1 peptides to purified HLA-DR molecules. Although a number of DR1-restricted epitopes were identified following Caf1 immunization, the response was biased toward a single immunodominant epitope near the C terminus of Caf1. In addition, potential promiscuous epitopes, including the immunodominant epitope, were identified by their ability to bind multiple common HLA alleles, with implications for the generation of multivalent vaccines against plague for use in humans.
Limited data are available on immunologic responses to primary H1N1 infection in patients with hematologic malignancies. We present a prospective, case-surveillance study of such patients with real-time polymerase chain reaction (RT-PCR) confirmed H1N1-influenza who presented to our institution between September 2009 and January 2010. Ninety-two patients presented with influenza-like symptoms, and 13 had H1N1 infection confirmed by RT-PCR, including 4 allogeneic stem cell transplant recipients (1 with acute myelogenous leukemia, 1 with chronic lymphoblastic leukemia [CLL], 1 with non-Hodgkin lymphoma, and 1 with chronic myelogenous leukemia), 5 patients with multiple myeloma following autologous stem cell transplantation, 1 patient with multiple myeloma perimobilization, 2 patients with NHL post chemotherapy, and 1 patient with CLL. All 13 patients required hospitalization. Six (43%) were admitted to the intensive care unit (ICU), of whom 4 (67%) died. We evaluated B cell and T cell responses to H1N1 infection prospectively in these patients compared with those in 4 otherwise healthy controls. Within 12 weeks of diagnosis, only 6 of 11 patients developed seropositive antibody titers as measured by hemagglutination-inhibition or microneutralization assays, compared with 4 of 4 controls. H1N1-specific T cells were detected in only 2 of 8 evaluable patients compared with 4 of 4 controls. H1N1-specific T cells were functional, capable of producing interferon ?, tumor necrosis factor ?, and CD107a mobilization. Furthermore, CD154 was up-regulated on CD4(+) T cells in 3 of 4 controls and 2 of 2 patients who had both B cell and T cell responses to H1N1. Post-H1N1 infection, 5 of 8 patients developed seasonal influenza-specific T cells, suggesting cross-reactivity induced by H1N1 infection. These data offer novel insights into humoral and cell-mediated immunologic responses to primary H1N1 infection.
Although subjects with inherited defects of the classical complement pathway component C2 are at increased risk of infection, there are few experimental data available on which bacterial pathogens they might be susceptible to. In order to investigate whether patients with inherited C2 deficiency may have increased susceptibility to Streptococcus pyogenes infection we have analysed opsonization with C3b/iC3b and phagocytosis of three different strains of S. pyogenes in serum from 8 C2(-/-) subjects using flow cytometry assays. Sera from patients with C2 deficiency had a markedly reduced ability to opsonise S. pyogenes with C3b/iC3b. In addition, phagocytosis of all three S. pyogenes strains was impaired in sera from C2(-/-) subjects. Both the reduced opsonisation with C3b/iC3b and phagocytosis in C2(-/-) sera were markedly improved by addition of exogenous C2 protein. Neutrophil dependent killing was also reduced, confirming the functional importance of C2 deficiency for immunity to S. pyogenes. Impaired opsonisation with C3b/iC3b and phagocytosis was not related to reduced recognition of the bacteria by antibody. These data suggest that patients with C2 deficiency are at increased risk of S. pyogenes infections.
There has been a long history of defining T cell epitopes to track viral immunity and to design rational vaccines, yet few data of this type exist for bacterial infections. Bacillus anthracis, the causative agent of anthrax, is both an endemic pathogen in many regions and a potential biological warfare threat. T cell immunity in naturally infected anthrax patients has not previously been characterized, which is surprising given concern about the ability of anthrax toxins to subvert or ablate adaptive immunity. We investigated CD4 T cell responses in patients from the Kayseri region of Turkey who were previously infected with cutaneous anthrax. Responses to B. anthracis protective Ag and lethal factor (LF) were investigated at the protein, domain, and epitope level. Several years after antibiotic-treated anthrax infection, strong T cell memory was detectable, with no evidence of the expected impairment in specific immunity. Although serological responses to existing anthrax vaccines focus primarily on protective Ag, the major target of T cell immunity in infected individuals and anthrax-vaccinated donors was LF, notably domain IV. Some of these anthrax epitopes showed broad binding to several HLA class alleles, but others were more constrained in their HLA binding patterns. Of specific CD4 T cell epitopes targeted within LF domain IV, one is preferentially seen in the context of bacterial infection, as opposed to vaccination, suggesting that studies of this type will be important in understanding how the human immune system confronts serious bacterial infection.
SpyCEP is a Streptococcus pyogenes protease that cleaves CXCL8/IL-8 and its activity is associated with human invasive disease severity. We investigated the role of SpyCEP in S. pyogenes necrotizing fasciitis and respiratory tract infection in mice using isogenic strains differing only in SpyCEP expression. SpyCEP cleaved human CXCL1, 2, 6 and 8 plus murine CXCL1 and 2 at a structurally conserved site. Mice were infected in thigh muscle with a strain of S. pyogenes that expresses a high level of SpyCEP, or with an isogenic non-SpyCEP expressing strain. SpyCEP expression by S. pyogenes hindered bacterial clearance from muscle, and enhanced bacterial spread, associated with cleavage of murine chemoattractant CXCL1. Mice were then infected with Lactococcus lactis strains that differed only in SpyCEP expression. In contrast to the parent L. lactis strain (lacks SpyCEP), which was avirulent when administered intramuscularly, infection with a strain that expressed SpyCEP heterologously led to dramatic systemic illness within 24 h, failure to clear bacteria from muscle and marked dissemination to other organs. In the upper airways, SpyCEP expression was required for survival of L. lactis but not S. pyogenes. However, dissemination of S. pyogenes to the lung was SpyCEP-dependent and was associated with evidence of chemokine cleavage. Taken together, the studies provide clear evidence that SpyCEP is necessary and sufficient for systemic bacterial dissemination from a soft tissue focus in this model and also underlies dissemination in the respiratory tract.
Neutrophil chemoattractant interleukin (IL)-8 is cleaved and inactivated by the Streptococcus pyogenes cell envelope protease SpyCEP. A range of clinical S. pyogenes strains of differing emm type demonstrated SpyCEP activity, although transcription of the SpyCEP gene cepA differed 1000-fold between isolates. Disruption of the 2-component regulatory system covR/S in pharyngeal isolates increased cepA transcription 100-fold; this finding is consistent with endogenous CovR/S-mediated repression of cepA being responsible for low SpyCEP expression in some S. pyogenes strains associated with pharyngitis. Among patients with invasive S. pyogenes infection, disease severity and outcome were associated with the SpyCEP activity of the isolate. Lethal invasive isolate H292 (emm81) expressed more cepA than did other tested isolates. This strain carried a unique covR mutation that impaired binding to the cepA promoter. CovR/S sequence comparison in other clinical isolates revealed community-wide dissemination of covS mutations but not covR mutations. The results highlight a potential hazard and underline the importance of continuing molecular epidemiological surveillance for community-wide dissemination of CovR/S mutant hyperinvasive strains.
Currently there is no licensed vaccine against the human pathogen Streptococcus pyogenes. The highly conserved IL-8 cleaving S. pyogenes cell envelope proteinase SpyCEP is surface expressed and is a potential vaccine candidate. A recombinant N-terminal part of SpyCEP (CEP) was expressed and purified. AntiCEP antibodies were found to neutralize the IL-8 cleaving activity of SpyCEP. CEP-immunized mice had reduced bacterial dissemination from focal S. pyogenes intramuscular infection and intranasal infection. We also identified a functional SpyCEP-homolog protease SeCEP, expressed by the equine pathogen Streptococcus equi, which was able to cleave both human and equine IL-8. CEP-immunized mice also demonstrated reduced bacterial dissemination from S. equi intramuscular infection. Therefore immunization against SpyCEP may provide protection against other streptococci species with homologous proteases.
Group A streptococci (GAS) cause a number of human diseases ranging from pharyngitis to necrotizing fasciitis. GAS are hypothesized to escape killing by either the immune system or beta lactam antibiotics by internalization into epithelial cells. A Tn917 library of transposon mutants was screened for capacity to invade and survive in human epithelial cells using a novel blood agar overlay method. Although the screen revealed that a majority of Tn917 insertions occurred within a 10 kb region of the genome, GAS genes identified as essential for internalization into epithelial cells included ABC transporters, and DNA maintenance proteins, and citrate metabolism enzymes, underlining the importance of adaptation to the intracellular environment.
Streptococcus pyogenes M/emm3 strains have been epidemiologically linked with enhanced infection severity and risk of streptococcal toxic shock syndrome (STSS), a syndrome triggered by superantigenic stimulation of T cells. Comparison of S. pyogenes strains causing STSS demonstrated that emm3 strains were surprisingly less mitogenic than other emm-types (emm1, emm12, emm18, emm28, emm87, emm89) both in vitro and in vivo, indicating poor superantigenic activity. We identified a 13 bp deletion in the superantigen smeZ gene of all emm3 strains tested. The deletion led to a premature stop codon in smeZ, and was not present in other major emm-types tested. Expression of a functional non-M3-smeZ gene successfully enhanced mitogenic activity in emm3 S. pyogenes and also restored mitogenic activity to emm1 and emm89 S. pyogenes strains where the smeZ gene had been disrupted. In contrast, the M3-smeZ gene with the 13 bp deletion could not enhance or restore mitogenicity in any of these S. pyogenes strains, confirming that M3-smeZ is non-functional regardless of strain background. The mutation in M3-smeZ reduced the potential for M3 S. pyogenes to induce cytokines in human tonsil, but not during invasive infection of superantigen-sensitive mice. Notwithstanding epidemiological associations with STSS and disease severity, emm3 strains have inherently poor superantigenicity that is explained by a conserved mutation in smeZ.
Streptococcus pyogenes is the causative agent of numerous diseases ranging from benign infections (pharyngitis and impetigo) to severe infections associated with high mortality (necrotizing fasciitis and bacterial sepsis). As with other bacterial infections, there is considerable interest in characterizing the contribution of interleukin-17A (IL-17A) responses to protective immunity. We here show significant il17a up-regulation by quantitative real-time PCR in secondary lymphoid organs, correlating with increased protein levels in the serum within a short time of S. pyogenes infection. However, our data offer an important caveat to studies of IL-17A responsiveness following antigen inoculation, because enhanced levels of IL-17A were also detected in the serum of sham-infected mice, indicating that inoculation trauma alone can stimulate the production of this cytokine. This highlights the potency and speed of innate IL-17A immune responses after inoculation and the importance of proper and appropriate controls in comparative analysis of immune responses observed during microbial infection.
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