Staphylococcal and Streptococcal Pyrogenic Toxins Involved in Toxic Shock Syndrome and Related Illnesses

Abstract

Toxic-shock syndrome (TSS) is an acute onset, multiorgan illness which resembles severe scarlet fever. The illness is caused by Staphylococcus aureus strains that express TSS toxin-1 (TSST-1), enterotoxin B, or enterotoxin C. TSST-1 is associated with menstrual TSS and approximately one-half of nonmenstrual cases; the other two toxins cause nonmenstrual cases, 47% and 3%, respectively. The three toxins are expressed in culture media under similar environmental conditions. These conditions may explain the association of certain tampons with menstrual TSS. Biochemically, the toxins are all relatively low molecular weight and fairly heat and protease stable. Enterotoxins B and C, share nearly 50% sequence ho-mology with streptococcal scarlet fever toxin A; they share no homology with TSST-1 despite sharing numerous biological properties. Numerous animal models for development of TSS have suggested mechanisms of toxin action, though the exact molecular action is not known. The toxins are all potent pyr-ogens, induce T lymphocyte proliferation, requiring interleukin 1 release from macrophages, suppress immunoglobulin production, enhance endotoxin shock, and enhance hypersensitiv-iry skin reactions. The genetic control of the toxins has been studied and suggests the exotoxins are variable traits. Some additional properties of TSS S. aureus which facilitate disease causation have been clarified.     

Detection of Staphylococcal Superantigenic Toxins by a CD69-Specific Cytofluorimetric Assay Measuring T-Cell Activation

The presence of staphylococcal superantigenic toxins in the supernatants of liquid cultures was detected by an easy and rapid method assessing the activation of T lymphocytes by cytofluorimetric measurement of CD69 expression. Staphylococcus aureus cells were grown in Eagle’s minimum essential medium supplemented with 5% heat-inactivated fetal calf serum. Supernatant fluids from all S. aureus strains producing superantigen-related toxins, including enterotoxins A to E, toxic shock syndrome toxin, and exfoliative toxins A and B, induced CD69 expression in a significantly higher number of T cells than a cutoff of 2%. This CD69 assay might be used for initial detection of superantigens from S. aureus strains isolated in the context of staphylococcal toxemia or related chronic human diseases such as atopic dermatitis or Kawasaki syndrome.     

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus Isolates from Patients Newly Identified as Nasal Carriers

We aimed to determine whether additional molecular and microbiological evaluations of methicillin-resistant Staphylococcus aureus (MRSA) isolated from patients newly identified as nasal carriers were useful for control strategies and whether longitudinal testing during the same or repeat hospitalization changed MRSA status. Nasal swabs from patients positive by Xpert MRSA PCR and not known to be colonized in the previous year were cultured for S. aureus. Isolates were tested for resistance to a variety of antibiotics, including high-level mupirocin resistance (HLMR) and low-level mupirocin resistance (LLMR) and the presence of genes mecA and mupA and those for Panton-Valentine leukocidin (PVL), USA300, and USA400. Repeat nasal screens during the 6-month study were tested for continued presence of MRSA. Among 130 patients, cultures revealed MRSA in 85 (65.4%), methicillin-susceptible S. aureus in 19 (14.6%), and no growth in 26 (20%). MRSA isolates were USA300 positive in 13/85 (15.3%) and LLMR in 8/85 (9.4%) patients. No isolates were HLMR or mupA positive. mecA dropout was detected in 9/130 (6.9%) patients. The rate of subsequent MRSA infections in USA300-positive versus -negative patients was not different. MRSA nasal status remained concordant in 69/70 (98.6%) patients who had follow-up testing. The findings do not support expanding MRSA surveillance to include routine detection of genes for USA300, PVL, or mupA, all of which were either of low frequency or not significantly associated with MRSA infection risk in our population of newly identified nasal carriers. Repeat nasal screening for MRSA during the same or subsequent hospitalizations over 6 months could also be deferred, reducing costs associated with screening.     

Newly Identified Enterovirus C Genotypes,Identified in the Netherlands through Routine Sequencing of All Enteroviruses Detected in Clinical Materials from 2008 to 2015

Enteroviruses (EVs) are a group of human and animal viruses that are capable of causing a variety of clinical syndromes. Different genotypes classified into species can be distinguished on the basis of sequence divergence in the VP1 capsid-coding region. Apparently new genotypes are discovered regularly, often as incidental findings in studies investigating respiratory syndromes or as part of poliovirus surveillance. Recently, some EVs have become recognized as significant respiratory pathogens, and a number of new genotypes belonging to species C have been identified. The circulation of these newly identified species C EVs, such as EV-C104, EV-C105, EV-C109, and EV-C117, nevertheless appears to be limited. In this report, we show the results of routine genotyping of all enteroviruses detected in our tertiary care hospital between January 2008 and April 2015. We detected 365 EVs belonging to 40 genotypes. Interestingly, several newly identified species C EVs were detected during the study period. Sequencing of the 5′ untranslated region (5′ UTR) of these viruses shows divergence in this region, which is a target region in many detection assays.     

A Newly Identified Missense Mutation in FARS2 Causes Autosomal‐Recessive Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous group of neurodegenerative disorders characterized by spasticity of the lower limbs due to pyramidal tract dysfunction. Here, we report that a missense homozygous mutation c.424G>T (p.D142Y) in the FARS2 gene, which encodes a mitochondrial phenylalanyl tRNA synthetase (mtPheRS), causes HSP in a Chinese consanguineous family by using combination of homozygous mapping and whole‐exome sequencing. Immunohistochemical experiments were performed showing that the FARS2 protein was highly expressed in the Purkinje cells of rat cerebellum. The aminoacylation activity of mtPheRS was severely disrupted by the p.D142Y substitution in vitro not only in the first aminoacylation step but also in the last transfer step. Taken together, our results indicate that a missense mutation in FARS2 contributes to HSP, which has the clinical significance of the regulation of tRNA synthetases in human neurodegenerative diseases.     

Use of Multiplex PCR To Detect Classical and Newly Described Pyrogenic Toxin Genes in Staphylococcal Isolates

Staphylococcus aureus may contain one or more genes that encode a variety of immunomodulatory pyrogenic toxins (PTs), including the staphylococcal enterotoxins and toxic shock syndrome toxin (TSST). The PTs interact with several cellular targets to produce disease, such as food poisoning and toxic shock syndrome. At present, nine serologically distinct enterotoxins and one immunoreactive form of TSST have been identified and characterized. As isolates of S. aureus are further assessed, it is anticipated that this number will increase. To facilitate screening, a multiplex PCR was designed to simultaneously determine which of these 10 currently known PT genes an individual S. aureus isolate possesses. We show here, using S. aureus isolates with characterized PT phenotypes, that this novel PCR technique reliably detects each of the known PTs in a single reaction.     

转录调控蛋白PrfA对两组新近发现的单核细胞增生李斯特菌基因的体外转录作用的研究

目的研究转录调控蛋白PrfA对两组新近发现的单核细胞增生李斯特菌基因的体外转录作用。方法利用本室近年来建立的体外转录系统,对两组基于转录基因组体内研究发现的5个可能的受PrfA不同调节的单核细胞增生李斯特菌基因进行了体外转录活性的研究。结果第一组中的hpt基因的体外转录活性受PrfA正调节,而其它4个基因既不被PrfA正调节也不被负调节。结论除hpt基因外,其它4个基因体外转录结果与体内实验不相一致,说明PrfA在体内可能通过复杂多样的非直接方式、或者还需要一些目前未知的因子来调控这些新近发现的基因的表达。     

Schistosoma mansoni Soluble Egg Antigens Enhance T Cell Responses to a Newly Identified HIV-1 Gag H-2b Epitope

Schistosome infection induces significant T helper type 2 (Th2) and anti-inflammatory immune responses and has been shown to negatively impact vaccine efficacy. Our goal was to determine if the administration of schistosome soluble egg antigens (SEA) would negatively influence the induction of cytotoxic T lymphocyte (CTL) and Th1-type T cell responses to an HIV candidate vaccine in the Th1-biased C57BL/6 mouse strain. Initial experiments failed, as we were unable to detect any response to the defined class I epitope for HIV-1 IIIB Gag. Therefore, we initiated an epitope mapping study to identify C57BL/6 (H-2^b) T cell epitopes in HIV-1 IIIB Gag in order to perform the experiments. This analysis defined two previously unreported minimal class I H-2^b and class II I-A^b epitopes for HIV-1 IIIB Gag. The newly defined HIV-1 IIIB Gag epitopes were used to evaluate the influence of SEA on the generation of CTL and Th1-type HIV-1 IIIB Gag responses. Surprisingly, in contrast to our hypothesis, we observed that the coadministration of SEA with a Listeria monocytogenes vector expressing HIV-1 IIIB Gag (Lm-Gag) led to a significantly increased frequency of gamma interferon (IFN-γ)-producing CD8⁺ and CD4⁺ T cells in C57BL/6 mice compared to mice immunized with Lm-Gag only. These observations suggest that SEA contains, in addition to Th2-type and immune-suppressive molecules, substances that can act with the Lm-Gag vaccine to increase CTL and Th1-type vaccine-specific immune responses.     

Molecular Epidemiology of Streptococcus pneumoniae Serogroup 6 Isolates from Fijian Children,Including Newly Identified Serotypes 6C and 6D

Multilocus sequence typing (MLST) was applied to all unique serotype 6C and 6D isolates and a random selection of serotype 6B and 6A isolates from nasopharyngeal swabs from Fijian children enrolled in a recent vaccine trial. The results suggest that Fijian serotype 6D has arisen independently from both serotypes 6A/C and 6B.Infection with Streptococcus pneumoniae is a leading cause of death in children worldwide (15, 19, 25). S. pneumoniae comprises 48 capsular serogroups containing more than 90 serotypes. Serogroup 6 classically consisted of serotypes 6A and 6B. The 7-valent conjugate vaccine (Prevnar; PCV7) includes the 6B antigen and confers some cross-protection against serotype 6A but not 6C (20, 22). Serotype 6C is important in carriage and invasive disease, and its prevalence has increased following widespread use of PCV7 (4, 5, 9, 10, 14, 16, 24).The existence of serotype 6D has been postulated (8) and was created experimentally (2), but naturally occurring isolates have not been identified (2, 8, 17) until recently, when we identified 14 naturally occurring serotype 6D isolates from nasopharyngeal swabs from Fijian children (11). Recently, two serotype 6D isolates have been found in Korean children (1).Initially, serotype 6C was postulated to have arisen from a single, possibly recent, evolutionary event in which serotype 6A wciN was replaced by serotype C wciN (21). Subsequent analyses, predominantly by multilocus sequence typing (MLST), have shown that 6C is genetically diverse, believed to be a consequence of multiple separate conversion events or a single event occurring sufficiently early in pneumococcal evolution (3, 4, 8, 9, 17).Serotype 6C is usually associated with clonal complexes (CCs) containing predominantly serotype 6A, and less commonly, 6B or non-serogroup-6 serotypes (3, 4, 9, 17). To date, MLST has been conducted on two serotype 6D isolates from Korea (ST282) (1), two from China (ST982 and ST4190), and one from Australia (ST4241) (http://spneumoniae.mlst.net). However, the molecular epidemiology of serotype 6D isolates is otherwise uncharacterized.In this study, we conducted MLST of serogroup 6 isolates to determine the genetic diversity and likely evolutionary origin of serotype 6C and 6D isolates.S. pneumoniae was isolated and identified as described elsewhere (18) from children aged 6 to 18 months participating in the Fiji Pneumococcal Project (FiPP) who had received 0, 1, 2, or 3 doses of PCV7 and 0 or 1 dose of 23-valent pneumococcal polysaccharide vaccine (PPV23) (23). Isolates were serotyped by a multiplex PCR-based reverse line blot (mPCR/RLB) assay and/or quellung reaction (before factor serum 6d was available), plus serogroup 6 serotype-specific PCR as previously described (11, 12).MLST was applied to all unique serotype 6C and 6D strains from the FiPP study (n = 52 and 24, respectively), of which 24 and 14 isolates, respectively, were included in our previous report (without ST results) (11). For comparison, we performed MLST on a subset of randomly selected serotype 6A (n = 16) and 6B (n = 17) isolates from the FiPP study.Fresh 18- to 24-h subcultures of S. pneumoniae isolates were suspended in nuclease-free water (Ambion), and genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen) per the manufacturer''s instructions. MLST was performed using primer pairs described by the Centers for Disease Control and Prevention (http://www.cdc.gov/ncidod/biotech/strep/alt-MLST-primers.htm) (aroE, recP, spi, xpt and ddl) or Enright and Spratt (6) (gdh and gki), except as described below.PCRs were conducted with 25-μl volumes containing approximately 5 ng of genomic DNA, 1 U of AmpliTaq DNA polymerase (Applied Biosystems), 1 × PCR buffer II (50 mM KCl, 10 mM Tris-HCl [pH 8.3]), 3.0 mM MgCl₂, 250 μM each deoxynucleoside triphosphate (dNTP), 0.5 μM forward primer, and 0.5 μM reverse primer (Sigma-Aldrich). PCR cycling conditions were a 5-min hold at 94°C, followed by 35 cycles at 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, and a final extension at 72°C for 5 min. Some isolates which produced no or small amounts of PCR product from spi (seven 6C and three 6D isolates) and/or recP (three 6C and one 6A isolate) were successfully amplified with primers described by Enright and Spratt (6) at an annealing temperature of 52°C in 4.5 mM MgCl₂.Amplicons were sequenced in both directions using capillary separation on the ABI 3730xl DNA analyzer with ABI BigDye Terminator labeling (version 3.1) (Australian Genome Research Facility) using the same primers as for amplification. Contiguous sequences were formed and edited using Sequencher 4.9 (Gene Codes Corporation).Allelic profiles and sequence types (STs) were obtained and compared to those of other isolates in the MLST database (http://spneumoniae.mlst.net). Relationships between STs were explored using eBURST version 3 software (Imperial College, London), which is available at the MLST website. For this study, a group was defined as two or more isolates which shared alleles at six of seven loci. Using this stringent definition, a group also defined a clonal complex (CC). Bootstrap analyses of the CC founder are also presented where appropriate.Generally, Fijian serogroup 6 isolates were highly clonal, with the predominant clone in each serotype representing >44% of the isolates. This is not surprising, given that all strains were isolated from children of similar ages over a relatively short period of time in a small, geographically isolated area.Serotype 6A isolates included STs 490 (7/16), 4778, 4779, 499, and 460 (Table ​(Table1).1). ST490 is predicted to be the CC founder (bootstrap value, 96%) and contains predominantly serotype 6A isolates in the MLST database. ST4778 and ST4779 are newly identified in this study and were not assigned to a CC. The only other ST499 isolate in the database (serotype 6A isolate from Finland) was also not assigned to a CC. ST460 is predicted to be a CC founder (bootstrap value, 88%). Most serotype 6A isolates (10/16) belonged to STs which contain only serotype 6A in the database (i.e., ST490, ST499, and ST460).

TABLE 1.

Distribution of STs and eBURST analysis for 109 serotype 6 isolates from Fijian children

Emetic action of staphylococcal enterotoxin A on weanling pigs.

Peroral and intraduodenal administration of staphylococcal enterotoxin A (SEA) to weanling pigs elicited an emetic response. Peroral administration of an emetic dose of SEA resulted in a single emetic episode occurring 90 to 180 min after dosing. Intraduodenal administration via a surgically implanted catheter of 100 or 150 micrograms of SEA resulted in multiple emetic episodes occurring 150 to 210 min after dosing, suggesting an intestinal site of action for SEA. The 50% emetic dose for perorally administered SEA was between 40 and 50 micrograms for 4.1- to 9.1-kg weanling pigs and 20 micrograms in 0.9- to 2.3-kg weanling pigs. Neurobehavioral responses, including alternating periods of drowsiness and restlessness, staggering, temporary loss of the righting reflex, and refusal to feed were also observed in pigs given an oral dose of SEA. Based on the demonstrated responsiveness of pigs to SEA, pigs should be considered suitable animal models for studies on the sites and mode of action of the toxin.     

UpaH Is a Newly Identified Autotransporter Protein That Contributes to Biofilm Formation and Bladder Colonization by Uropathogenic Escherichia coli CFT073

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.Urinary tract infections (UTIs) are among the most common infectious diseases of humans. It is estimated that 40 to 50% of adult healthy women have experienced at least one UTI episode in their lifetime, and there is a tendency for these infections to become chronic due to a high rate of recurrence. Almost all patients with an indwelling urinary catheter for 30 days or longer develop catheter-associated UTI, which accounts for 40% of all nosocomial infections (13).Uropathogenic Escherichia coli (UPEC) is the cause of the majority (>80%) of UTIs in humans and one of the most common sources of Gram-negative bacteremia in hospitalized patients. The ability of UPEC to colonize the urinary tract and cause disease involves adhesins (e.g., type 1 and P fimbriae), toxins (e.g., hemolysin), and iron acquisition systems that utilize siderophores (e.g., enterobactin, salmochelin, and aerobactin) (31, 60). Adherence to the urinary tract epithelium enables bacteria to resist the hydrodynamic forces of urine flow, to trigger host and bacterial cell signaling pathways, and to establish infection. Among adhesins, P and type 1 fimbriae correlate strongly with uropathogenesis and mediate binding to specific receptors in the urinary tract (7, 39, 47, 54, 62, 63). Both P and type 1 fimbriae recognize their receptor targets by virtue of organelle tip-located adhesins, PapG and FimH, respectively (27, 33).In addition to fimbrial adhesins, a number of autotransporter (AT) proteins associated with virulence have been characterized for UPEC. These include the secreted toxin Sat (17, 18), the phase-variable outer membrane protein antigen 43 (Ag43) (57), and the surface-located trimeric AT protein UpaG (59). AT proteins are unique in that their sequence is sufficient to direct their transport across the bacterial membrane system and final routing of a variable passenger (α) domain to the cell surface. AT proteins are generally highly similar with respect to the structure of the translocation (β) domain that assists the transport of the α-domain across the outer membrane, whereas they differ substantially in their α-domain, which determines the unique functional characteristics of AT proteins (23, 24). Once at the bacterial surface, the α-domain may be processed and released into the extracellular surroundings (e.g., Pet and EspP), or it may be cleaved but remain in contact with the cell surface via noncovalent interactions with the β-domain (e.g., Ag43 and AIDA). Thus, AT proteins have diverse functions, ranging from cell-associated adhesins to secreted toxins (24).Ten putative AT-encoding genes have been identified in the sequenced genome of the prototype UPEC strain CFT073 (45). In this study, we identified and characterized a new AT-encoding gene from CFT073, which we have named upaH. The upaH gene was not identified in the original analysis of the CFT073 genome sequence due to a sequence misassembly within a highly repetitive region. We show that upaH encodes a large cell surface-located AT protein that contributes to biofilm formation and colonization of the mouse urinary tract in coinfection experiments.     

Antigenic Cross-Reactivity of Staphylococcal Enterotoxins

The antigenic cross-reactivity of staphylococcal enterotoxins types A, B, and C was assessed using anti-A and anti-B antitoxins in the solid-phase radioimmunoassay test. Heterologous reactions were observed. At the 33% inhibition level, B was 18,000 and 5,400 times more effective as an inhibitor in its homologous system than were the heterologous enterotoxins A and C, respectively. Similarly, in the A system, A enterotoxin was 55,000 and 25,000 times more effective than were B and C toxins, respectively, in inhibiting A-anti-A reactions.     

Further Characterization of Staphylococcal Gamma-Hemolysin

It was confirmed that staphylococcal gamma-hemolysin is composed of two separate proteins (gamma-lysin components I and II) which act synergistically. The molecular weights of the two components, determined by gel filtration, are 29,000 and 26,000, respectively, and their isoelectric points, determined by isoelectric focusing, are at pH 9.8 and 9.9. Both components are susceptible to the action of Pronase and subtilisin. A wide range of lipids, some in minute amounts, are capable of inhibiting the hemolytic activity of gamma-lysin.     

Involvement of Enterotoxins G and I in Staphylococcal Toxic Shock Syndrome and Staphylococcal Scarlet Fever

We investigated the involvement of the recently described staphylococcal enterotoxins G and I in toxic shock syndrome. We reexamined Staphylococcus aureus strains isolated from patients with menstrual and nonmenstrual toxic shock syndrome (nine cases) or staphylococcal scarlet fever (three cases). These strains were selected because they produced none of the toxins known to be involved in these syndromes (toxic shock syndrome toxin 1 and enterotoxins A, B, C, and D), enterotoxin E or H, or exfoliative toxin A or B, despite the fact that superantigenic toxins were detected in a CD69-specific flow cytometry assay measuring T-cell activation. Sets of primers specific to the enterotoxin G and I genes (seg and sei, respectively) were designed and used for PCR amplification. All of the strains were positive for seg and sei. Sequence analysis confirmed that the PCR products, corresponded to the target genes. We suggest that staphylococcal enterotoxins G and I may be capable of causing human staphylococcal toxic shock syndrome and staphylococcal scarlet fever.     

Production of Antitoxins to Two Toxins of Clostridium difficile and Immunological Comparison of the Toxins by Cross-Neutralization Studies

We prepared antitoxins specific for each of two toxins of Clostridium difficile and used these to demonstrate that the toxins are immunologically distinct.