Staphylococcus aureus Toxins: Promoter or Handicap during Infection

Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient’s study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S. aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections. Full article

Staphylococcus aureus generates and releases extracellular vesicles (EVs) that package cytosolic, cell-wall associated, and membrane proteins, as well as glycopolymers and exoproteins, including alpha hemolysin, leukocidins, phenol-soluble modulins, superantigens, and enzymes. S. aureus EVs, but not EVs from pore-forming toxin-deficient strains, were cytolytic for a variety of mammalian cell types, but EV internalization was not essential for cytotoxicity. Because S. aureus is subject to various environmental stresses during its encounters with the host during infection, we assessed how these exposures affected EV production in vitro. Staphylococci grown at 37 °C or 40 °C did not differ in EV production, but cultures incubated at 30 °C yielded more EVs when grown to the same optical density. S. aureus cultivated in the presence of oxidative stress, in iron-limited media, or with subinhibitory concentrations of ethanol, showed greater EV production as determined by protein yield and quantitative immunoblots. In contrast, hyperosmotic stress or subinhibitory concentrations of erythromycin reduced S. aureus EV yield. EVs represent a novel S. aureus secretory system that is affected by a variety of stress responses and allows the delivery of biologically active pore-forming toxins and other virulence determinants to host cells. Full article

Staphylococcus aureus is a major human pathogen, inducing several infections ranging from the benign to the life-threatening, such as necrotising pneumonia. S. aureus is capable of producing a great variety of virulence factors, such as bicomponent pore-forming leucocidin, which take part in the physiopathology of staphylococcal infection. In necrotising pneumonia, Panton–Valentine leucocidin (PVL) induces not only lung injury and necrosis, but also leukopenia, regarded as a major factor of a poor prognosis. The aim of the present study was to evaluate the effect of bicomponent pore-forming leucocidin, PVL and gamma haemolysin on bone marrow leucocytes, to better understand the origin of leukopenia. Using multi-parameter cytometry, the expression of leucocidin receptors (C5aR, CXCR1, CXCR2, and CCR2) was assessed and toxin-induced lysis was measured for each bone marrow leucocyte population. We observed that PVL resulted in myeloid-derived cells lysis according to their maturation and their C5aR expression; it also induced monocytes lysis according to host susceptibility. Haemolysin gamma A, B, and C (HlgABC) displayed cytotoxicity to monocytes and natural killer cells, hypothetically through CXCR2 and CXCR1 receptors, respectively. Taken together, the data suggest that PVL and HlgABC can lyse bone marrow leucocytes. Nevertheless, the origin of leukopenia in severe staphylococcal infection is predominantly peripheral, since immature cells stay insensitive to leucocidins. Full article

Staphylococcal toxic shock syndrome is a potentially lethal illness attributed to superantigens produced by Staphylococcus aureus, in particular toxic shock syndrome toxin 1 (TSST-1), but staphylococcal enterotoxins (SEs) are also implicated. The genes encoding these important toxins are carried on mobile genetic elements, and the regulatory networks controlling expression of these toxins remain relatively unexplored. We show here that the highly conserved ClpXP protease stimulates transcription of tst (TSST-1), sec (SEC), and sed (SED) genes in the prototypical strains, SA564 and RN4282. In the wild-type cells, the post-exponential upregulation of toxin gene transcription was proposed to occur via RNAIII-mediated downregulation of the Rot repressor. Contradictive to this model, we showed that the post-exponential induction of tst, sed, and sec transcription did not occur in cells devoid of ClpXP activity, despite the Rot level being diminished. To identify transcriptional regulators with a changed expression in cells devoid of ClpXP activity, RNA sequencing was performed. The RNAseq analysis revealed a number of global virulence regulators that might act downstream of ClpXP, to control expression of tst and other virulence genes. Collectively, the results extend our understanding of the complex transcriptional regulation of the tst, sed, and sec genes. Full article

The pore-forming cytotoxin α-hemolysin, or Hla, is a critical Staphylococcus aureus virulence factor that promotes infection by causing tissue damage, excessive inflammation, and lysis of both innate and adaptive immune cells, among other cellular targets. In this study, we asked whether a virus-like particle (VLP)-based vaccine targeting Hla could attenuate S. aureus Hla-mediated pathogenesis. VLPs are versatile vaccine platforms that can be used to display target antigens in a multivalent array, typically resulting in the induction of high titer, long-lasting antibody responses. In the present study, we describe the first VLP-based vaccines that target Hla. Vaccination with either of two VLPs displaying a 21 amino-acid linear neutralizing domain (LND) of Hla protected both male and female mice from subcutaneous Hla challenge, evident by reduction in lesion size and neutrophil influx to the site of intoxication. Antibodies elicited by VLP-LND vaccination bound both the LND peptide and the native toxin, effectively neutralizing Hla and preventing toxin-mediated lysis of target cells. We anticipate these novel and promising vaccines being part of a multi-component S. aureus vaccine to reduce severity of S. aureus infection. Full article

Staphylococcus aureus and cystic fibrosis (CF) are closely interlinked. To date, however, the impact of S. aureus culture in CF airways on lung function and disease progression has only been elucidated to a limited degree. This analysis aims to identify bacterial factors associated to clinical deterioration. Data were collected during an observational prospective multi-center study following 195 patients from 17 centers. The average follow-up time was 80 weeks. S. aureus isolates (n = 3180) were scanned for the presence of 25 virulence genes and agr-types using single and multiplex PCR. The presence of specific virulence genes was not associated to clinical deterioration. For the agr-types 1 and 4, however, a link to the subjects’ clinical status became evident. Furthermore, a significant longitudinal decrease in the virulence gene quantity was observed. Analyses of the plasticity of the virulence genes revealed significantly increased plasticity rates in the presence of environmental stress. The results suggest that the phylogenetic background defines S. aureus pathogenicity rather than specific virulence genes. The longitudinal loss of virulence genes most likely reflects the adaptation process directed towards a persistent and colonizing rather than infecting lifestyle. Full article

Staphylococcal bi-component pore-forming toxins, also known as leukocidins, target and lyse human phagocytes in a receptor-dependent manner. S-components of the leukocidins Panton-Valentine leukocidin (PVL), γ-haemolysin AB (HlgAB) and CB (HlgCB), and leukocidin ED (LukED) specifically employ receptors that belong to the class of G-protein coupled receptors (GPCRs). Although these receptors share a common structural architecture, little is known about the conserved characteristics of the interaction between leukocidins and GPCRs. In this study, we investigated host cellular pathways contributing to susceptibility towards S. aureus leukocidin cytotoxicity. We performed a genome-wide CRISPR/Cas9 library screen for toxin-resistance in U937 cells sensitized to leukocidins by ectopic expression of different GPCRs. Our screen identifies post-translational modification (PTM) pathways involved in the sulfation and sialylation of the leukocidin-receptors. Subsequent validation experiments show differences in the impact of PTM moieties on leukocidin toxicity, highlighting an additional layer of refinement and divergence in the staphylococcal host-pathogen interface. Leukocidin receptors may serve as targets for anti-staphylococcal interventions and understanding toxin-receptor interactions will facilitate the development of innovative therapeutics. Variations in the genes encoding PTM pathways could provide insight into observed differences in susceptibility of humans to infections with S. aureus. Full article

Toxins produced by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) contribute to virulence. We developed a statistical approach to determine an optimum sequence of antimicrobials to treat CA-MRSA infections based on an antimicrobial’s ability to reduce virulence. In an in vitro pharmacodynamic hollow fiber model, expression of six virulence genes (lukSF-PV, sek, seq, ssl8, ear, and lpl10) in CA-MRSA USA300 was measured by RT-PCR at six time points with or without human-simulated, pharmacokinetic dosing of five antimicrobials (clindamycin, minocycline, vancomycin, linezolid, and trimethoprim/sulfamethoxazole (SXT)). Statistical modeling identified the antimicrobial causing the greatest decrease in virulence gene expression at each time-point. The optimum sequence was SXT at T0 and T4, linezolid at T8, and clindamycin at T24–T72 when lukSF-PV was weighted as the most important gene or when all six genes were weighted equally. This changed to SXT at T0–T24, linezolid at T48, and clindamycin at T72 when lukSF-PV was weighted as unimportant. The empirical p-value for each optimum sequence according to the different weights was 0.001, 0.0009, and 0.0018 with 10,000 permutations, respectively, indicating statistical significance. A statistical method integrating data on change in gene expression upon multiple antimicrobial exposures is a promising tool for identifying a sequence of antimicrobials that is effective in sustaining reduced CA-MRSA virulence. Full article

Staphylococcus aureus is a facultative pathogenic bacterium that colonizes the nasopharyngeal area of healthy individuals, but can also induce severe infection, such as pneumonia. Pneumonia caused by mono- or superinfected S. aureus leads to high mortality rates. To establish an infection, S. aureus disposes of a wide variety of virulence factors, which can vary between clinical isolates. Our study aimed to characterize pneumonia isolates for their virulent capacity. For this, we analyzed isolates from colonization, pneumonia due to S. aureus, and pneumonia due to S. aureus/influenza virus co-infection. A total of 70 strains were analyzed for their virulence genes and the host–pathogen interaction was analyzed through functional assays in cell culture systems. Strains from pneumonia due to S. aureus mono-infection showed enhanced invasion and cytotoxicity against professional phagocytes than colonizing and co-infecting strains. This corresponded to the high presence of cytotoxic components in pneumonia strains. By contrast, strains obtained from co-infection did not exhibit these virulence characteristics and resembled strains from colonization, although they caused the highest mortality rate in patients. Taken together, our results underline the requirement of invasion and toxins to cause pneumonia due to S. aureus mono-infection, whereas in co-infection even low-virulent strains can severely aggravate pneumonia. Full article

Chronic rhinosinusitis (CRS) is thought to be a multifactorial disease that includes a direct involvement of bacteria that trigger inflammation and contribute to CRS pathogenesis. Staphylococcus aureus infection and persistence is associated with chronic rhinosinusitis (CRS), and it may be particularly relevant in the form with nasal polyps (CRSwNP). The large array of exotoxins deployed by S. aureus is instrumental for the bacterium to warrant its infection and dissemination in different human body districts. Here, we analyze the common Th2 environment in CRSwNP and prospect a possible dynamic role played by S. aureus leukocidins in promoting this chronic inflammation, considering leukocidin ED (LukED) as a strong prototype candidate worth of therapeutic investigation. CCR5 is an essential target for LukED to exert its cytotoxicity towards T cells, macrophages and dendritic cells. Therefore, CCR5 blockade might be an interesting therapeutic option for CRS and, more specifically, persistent and relapsing CRSwNP. In this perspective, the arsenal of CCR5 antagonists being developed to inhibit HIV-1 entry (CCR5 being the major HIV-1 co-receptor) could be easily repurposed for CRS therapeutic investigation. Finally, direct targeting of LukED by neutralizing antibodies could represent an important additional solution to S. aureus infection. Full article

Staphylococcal enterotoxins are the most common cause of foodborne intoxications (staphylococcal food poisoning) and cause a wide range of diseases. With at least six variants staphylococcal enterotoxin C (SEC) stands out as particularly diverse amongst the 25 known staphylococcal enterotoxins. Some variants present unique and even host-specific features. Here, we review the role of SEC in human and animal health with a particular focus on its role as a causative agent for foodborne intoxications. We highlight structural features unique to SEC and its variants, particularly, the emetic and superantigen activity, as well as the roles of SEC in mastitis and in dairy products. Information about the genetic organization as well as regulatory mechanisms including the accessory gene regulator and food-related stressors are provided. Full article

Staphylococcus aureus is a metabolically flexible pathogen that causes infection in diverse settings. An array of virulence factors, including the secreted toxins, enables S. aureus to colonize different environmental niches and initiate infections by any of several discrete pathways. During these infections, both S. aureus and host cells compete with each other for nutrients and remodel their metabolism for survival. This metabolic interaction/crosstalk determines the outcome of the infection. The reprogramming of metabolic pathways in host immune cells not only generates adenosine triphosphate (ATP) to meet the cellular energy requirements during the infection process but also activates antimicrobial responses for eventual bacterial clearance, including cell death pathways. The selective pressure exerted by host immune cells leads to the emergence of bacterial mutants adapted for chronicity. These host-adapted mutants are often characterized by substantial changes in the expression of their own metabolic genes, or by mutations in genes involved in metabolism and biofilm formation. Host-adapted S. aureus can rewire or benefit from the metabolic activities of the immune cells via several mechanisms to cause persistent infection. In this review, we discuss how S. aureus activates host innate immune signaling, which results in an immune metabolic pressure that shapes S. aureus metabolic adaptation and determines the outcome of the infection. Full article

Staphylococcus aureus constitutes a major food-borne pathogen, as well as one of the main causative agents of mastitis in dairy ruminants. This pathogen can produce a variety of extracellular toxins; these include the shock syndrome toxin 1 (TSST-1), exfoliative toxins, staphylococcal enterotoxins (SE), hemolysins, and leukocidins. S. aureus expresses many virulence proteins, involved in evading the host defenses, hence facilitating microbial colonization of the mammary glands of the animals. In addition, S. aureus exotoxins play a role in the development of both skin infections and mastitis. Indeed, if these toxins remain in dairy products for human consumption, they can cause staphylococcal food poisoning (SFP) outbreaks. As a result, there is a need for procedures to identify the presence of exotoxins in human food, and the methods used must be fast, sensitive, reliable, and accurate. It is also essential to determine the best medical therapy for human patients suffering from S. aureus infections, as well as establishing the relevant veterinary treatment for infected ruminants, to avoid economic losses in the dairy industry. This review summarizes the role of S. aureus toxins in the development of mastitis in ruminants, their negative effects in the food and dairy industries, and the different methods used for the identification of these toxins in food destined for human consumption. Full article

Staphylococcus aureus is a Gram-positive pathogen capable of infecting nearly every vertebrate organ. Among these tissues, invasive infection of bone (osteomyelitis) is particularly common and induces high morbidity. Treatment of osteomyelitis is notoriously difficult and often requires debridement of diseased bone in conjunction with prolonged antibiotic treatment to resolve infection. During osteomyelitis, S. aureus forms characteristic multicellular microcolonies in distinct niches within bone. Virulence and metabolic responses within these multicellular microcolonies are coordinated, in part, by quorum sensing via the accessory gene regulator (agr) locus, which allows staphylococcal populations to produce toxins and adapt in response to bacterial density. During osteomyelitis, the Agr system significantly contributes to dysregulation of skeletal homeostasis and disease severity but may also paradoxically inhibit persistence in the host. Moreover, the Agr system is subject to complex crosstalk with other S. aureus regulatory systems, including SaeRS and SrrAB, which can significantly impact the progression of osteomyelitis. The objective of this review is to highlight Agr regulation, its implications on toxin production, factors that affect Agr activation, and the potential paradoxical influences of Agr regulation on disease progression during osteomyelitis. Full article