Cathelicidin antimicrobial peptide expression is not induced or required for bacterial clearance during salmonella enterica infection of human monocyte-derived macrophages.

Salmonella enterica serovar Typhimurium is able to resist antimicrobial peptide killing by induction of the PhoP-PhoQ and PmrA-PmrB two-component systems and the lipopolysaccharide (LPS) modifications they mediate. Murine cathelin-related antimicrobial peptide (CRAMP) has been reported to inhibit S. Typhimurium growth in vitro and in vivo. We hypothesize that infection of human monocyte-derived macrophages (MDMs) with Salmonella enterica serovar Typhi and S. Typhimurium will induce human cathelicidin antimicrobial peptide (CAMP) production, and exposure to LL-37 (processed, active form of CAMP/hCAP18) will lead to upregulation of PmrAB-mediated LPS modifications and increased survival in vivo. Unlike in mouse macrophages, in which CRAMP is upregulated during infection, camp gene expression was not induced in human MDMs infected with S. Typhi or S. Typhimurium. Upon infection, intracellular levels of ΔphoPQ, ΔpmrAB, and PhoP(c) S. Typhi decreased over time but were not further inhibited by the vitamin D(3)-induced increase in camp expression. MDMs infected with wild-type (WT) S. Typhi or S. Typhimurium released similar levels of proinflammatory cytokines; however, the LPS modification mutant strains dramatically differed in MDM-elicited cytokine levels. Overall, these findings indicate that camp is not induced during Salmonella infection of MDMs nor is key to Salmonella intracellular clearance. However, the cytokine responses from MDMs infected with WT or LPS modification mutant strains differ significantly, indicating a role for LPS modifications in altering the host inflammatory response. Our findings also suggest that S. Typhi and S. Typhimurium elicit different proinflammatory responses from MDMs, despite being capable of adding similar modifications to their LPS structures.

Proinflammatory exoprotein characterization of toxic shock syndrome Staphylococcus aureus.

Pulsed-field gel electrophoresis (PFGE) clonal type USA200 is the most widely disseminated Staphylococcus aureus colonizer of the nose and is a major cause of toxic shock syndrome (TSS). Exoproteins derived from these organisms have been suggested to contribute to their colonization and causation of human diseases but have not been well-characterized. Two representative S. aureus USA200 isolates, MNPE (α-toxin positive) and CDC587 (α-toxin mutant), isolated from pulmonary post-influenza TSS and menstrual vaginal TSS, respectively, were evaluated. Biochemical, immunobiological, and cell-based assays, including mass spectrometry, were used to identify key exoproteins derived from the strains that are responsible for proinflammatory and cytotoxic activity on human vaginal epithelial cells. Exoproteins associated with virulence were produced by both strains, and cytolysins (α-toxin and γ-toxin), superantigens, and proteases were identified as the major exoproteins, which caused epithelial cell inflammation and cytotoxicity. Exoprotein fractions from MNPE were more proinflammatory and cytotoxic than those from CDC587 due to high concentrations of α-toxin. CDC587 produced a small amount of α-toxin, despite the presence of a stop codon (TAG) at codon 113. Additional exotoxin identification studies of USA200 strain [S. aureus MN8 (α-toxin mutant)] confirmed that MN8 also produced low levels of α-toxin despite the same stop codon. The differences observed in virulence factor profiles of two USA200 strains provide insight into environmental factors that select for specific virulence factors. Cytolysins, superantigens, and proteases were identified as potential targets, where toxin neutralization may prevent or diminish epithelial damage associated with S. aureus.

Salmonella-regulated lipopolysaccharide modifications.

Salmonella enterica are Gram-negative enteric pathogens that cause typhoid fever and gastroenteritis in humans. Many bacteria, including Salmonella, use signal transduction cascades such as two-component regulatory systems to detect and respond to stimuli in the local microenvironment. During infection, environmental sensing allows bacteria to regulate gene expression to evade host immune defenses and thrive in vivo. Activation of the Salmonella two-component regulatory systems PhoP-PhoQ and PmrA-PmrB and the RcsC-RcsD-RcsB phosphorylay by specific environmental signals in the intestine and within host cells leads to several lipopolysaccharide modifications that promote bacterial survival, cationic antimicrobial peptide resistance and virulence. Many pathogens encode orthologs to Salmonella two-component regulatory systems and also modify the lipopolysaccharide to escape killing by the host immune response. However, these organisms often regulate their virulence genes, including those responsible for lipopolysaccharide modification, in ways that differ from Salmonella. Further examination of bacterial virulence gene regulation and lipopolysaccharide modifications may lead to improved antimicrobial therapies and vaccines.

Secreted virulence factor comparison between methicillin-resistant and methicillin-sensitive Staphylococcus aureus, and its relevance to atopic dermatitis.

Community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains have emerged as serious health threats in the last 15 years. They are associated with large numbers of atopic dermatitis skin and soft tissue infections, but when they originate from skin and mucous membranes, have the capacity to produce sepsis and highly fatal pulmonary infections characterized as necrotizing pneumonia, purpura fulminans, and postviral toxic shock syndrome. This review is a discussion of the emergence of 3 major CA-MRSA organisms, designated CA-MRSA USA400, followed by USA300, and most recently USA200. CA-MRSA USA300 and USA400 isolates and their methicillin-sensitive counterparts (community-associated methicillin-sensitive S aureus) typically produce highly inflammatory cytolysins alpha-toxin, gamma-toxin, delta-toxin (as representative of the phenol soluble modulin family of cytolysins), and Panton Valentine leukocidin. USA300 isolates produce the superantigens enterotoxin-like Q and a highly pyrogenic deletion variant of toxic shock syndrome toxin 1 (TSST-1), whereas USA400 isolates produce the superantigens staphylococcal enterotoxin B or staphylococcal enterotoxin C. USA200 CA-MRSA isolates produce small amounts of cytolysins but produce high levels of TSST-1. In contrast, their methicillin-sensitive S aureus counterparts produce various cytolysins, apparently in part dependent on the niche occupied in the host and levels of TSST-1 expressed. Significant differences seen in production of secreted virulence factors by CA-MRSA versus hospital-associated methicillin-resistant S aureus and community-associated methicillin-sensitive S aureus strains appear to be a result of the need to specialize as the result of energy drains from both virulence factor production and methicillin resistance.

Staphylococcal superantigens cause lethal pulmonary disease in rabbits.

BACKGROUND: The Centers for Disease Control and Prevention (CDC) and others reported that methicillin-resistant S. aureus (MRSA) are significant causes of serious human infections, including pulmonary illnesses. We investigated the role played by superantigens in lung-associated lethal illness in rabbits. METHODS: A rabbit model was established to investigate the potential role played by superantigens, staphylococcal enterotoxin B (SEB), staphylococcal enterotoxin C (SEC), and toxic shock syndrome toxin-1 (TSST-1). Rabbits received intrabronchial community-associated (CA) MRSA strains USA200 (TSST-1(+)), MW2 (SEC(+)), c99-529 (SEB(+)), or purified superantigens. Some rabbits were preimmunized against superantigens or treated with soluble high-affinity T cell receptors (Vß-TCR) to neutralize SEB and then challenged intrabronchially with CA-MRSA or superantigens. RESULTS: Rabbits challenged with CA-MRSA or superantigens developed fatal, pulmonary illnesses. Animals preimmunized against purified superantigens, or treated passively with Vß-TCRs and then challenged with CA-MRSA or superantigens, survived. Lung histological analysis indicated that nonimmune animals developed lesions consistent with necrotizing pneumonia after challenge with CA-MRSA or purified superantigens. Superantigen-immune animals or animals treated with soluble Vß-TCRs did not develop pulmonary lesions. CONCLUSIONS: Superantigens contribute to lethal pulmonary illnesses due to CA-MRSA; preexisting immunity to superantigens prevents lethality. Administration of high-affinity Vß-TCR with specificity for SEB to nonimmune animals protects from lethal pulmonary illness resulting from SEB(+) CA-MRSA and SEB.

Glycerol monolaurate inhibits Candida and Gardnerella vaginalis in vitro and in vivo but not Lactobacillus.

We investigated the effects of glycerol monolaurate (GML) on Lactobacillus, Candida, and Gardnerella vaginalis human vaginal microflora. Our previous work demonstrated that 6 months of GML treatment vaginally does not alter lactobacillus counts in monkeys. Candida and G. vaginalis are commonly associated with vaginal infections in women, many becoming chronic or recurrent. In vitro growth inhibition studies determined the effects of GML (0 to 500 microg/ml) against multiple Candida species and G. vaginalis. A randomized, double-blind study investigated the effects of GML on vaginal microflora Lactobacillus, Candida, and G. vaginalis in colonized or infected women (n=36). Women self-administered intravaginal gels containing 0% (n=14), 0.5% (n=13), or 5% (n=9) GML every 12 h for 2 days. Vaginal swabs were collected before and immediately after the first gel administration and 12 h after the final gel administration. Swabs were tested for Lactobacillus, Candida, G. vaginalis, and GML. In vitro GML concentrations of 500 microg/ml were candicidal for all species tested, while a concentration of 10 microg/ml was bactericidal for G. vaginalis. Control and GML gels applied vaginally in women did not alter vaginal pH or Lactobacillus counts. Control gels reduced G. vaginalis counts but not Candida counts, whereas GML gels reduced both Candida and G. vaginalis. No adverse events were reported by participating women. GML is antimicrobial for Candida and G. vaginalis in vitro. Vaginal GML gels in women do not affect Lactobacillus negatively but significantly reduce Candida and G. vaginalis.

Extreme pyrexia and rapid death due to Staphylococcus aureus infection: analysis of 2 cases.

We describe unusual Staphylococcus aureus infections in 2 patients. The infections were characterized by extreme pyrexia and rapid death. Both causative organisms produced a deletion mutant form of toxic shock syndrome toxin-1 and variant enterotoxin C, which may have caused pyrexia and death.

Reduction in Staphylococcus aureus growth and exotoxin production and in vaginal interleukin 8 levels due to glycerol monolaurate in tampons.

BACKGROUND: Staphylococcal menstrual toxic shock syndrome depends on vaginal production of exotoxins. Glycerol monolaurate (GML) inhibits Staphylococcus aureus exotoxin production in vitro. The purpose of this study was to determine whether GML, as a tampon fiber finish, inhibits production of exotoxins and the cytokine interleukin 8 (IL-8) during normal tampon use. METHODS: On day 2 of menstruation, when vaginal S. aureus counts are high in colonized women, study participants exchanged their own preferred tampons, after wearing them for 2-6 h, for study tampons with or without GML (assigned randomly and blindly), which they then wore for 4-6 h. The women's own tampons and the study tampons with or without GML were assayed for S. aureus, the exotoxins toxic shock syndrome toxin 1 and alpha-toxin, and IL-8. RESULTS: A total of 225 women completed the study. S. aureus was present in the tampons of 41 women (18%). Lower numbers of S. aureus and the exotoxins were detected in study tampons with or without GML than in women's own tampons; lower amounts of the exotoxins were present in study tampons with GML than study tampons without GML. The IL-8 level was lower in tampons from women without vaginal S. aureus compared with women with S. aureus and was lower in study tampons with GML than in study tampons without GML. CONCLUSIONS: Tampons that contain GML reduce S. aureus exotoxin production. S. aureus increases vaginal IL-8 levels, and GML reduces production of this proinflammatory cytokine. These results suggest that GML added to tampons provides additional safety relative to menstrual toxic shock syndrome as well as benefits for vaginal health generally, thus supporting the addition of GML to tampons.

Superantigen profile of Staphylococcus aureus isolates from patients with steroid-resistant atopic dermatitis.

BACKGROUND: Superantigens induce skin inflammatory responses in atopic dermatitis, which is commonly associated with Staphylococcus aureus infection. T cells activated in vitro by superantigens become steroid resistant. The objective was to assess the superantigen profiles of S. aureus isolates from patients with steroid-resistant atopic dermatitis. METHODS: We compared the superantigen-production capability of S. aureus isolates from 78 patients with steroid-resistant atopic dermatitis (group 1) with that of 30 vaginal isolates from healthy women (group 2) and 22 isolates from a general population of patients with atopic dermatitis (group 3). Polymerase chain reaction with primers for superantigens, combined with selected antibody testing, was used to analyze the presence of toxic shock syndrome toxin 1, staphylococcal enterotoxins, and enterotoxin-like superantigens. RESULTS: S. aureus isolates from group 1 had a statistically significant difference in superantigen profile, compared with the profiles of group 2 and group 3 isolates. Group 2 isolates were similar in profile to group 3 isolates, with 4 and 5 superantigens per isolate, respectively. In contrast, group 1 isolates produced a mean of 8 superantigens each (P<<.001, for comparison with group 2 or group 3). These group 1 isolates were more likely to produce the 3 major toxic shock syndrome-associated superantigens (toxic shock syndrome toxin 1, staphylococcal enterotoxin B, and staphylococcal enterotoxin C) and to produce unusual combinations of superantigens (e.g., toxic shock syndrome toxin 1 and staphylococcal enterotoxin B). CONCLUSIONS: S. aureus isolates from patients with steroid-resistant atopic dermatitis appear to be selected on the basis of greater production of superantigens, compared with that of isolates from control groups. Superantigens may offer selective advantages for colonization of patients.

Glycerol monolaurate does not alter rhesus macaque (Macaca mulatta) vaginal lactobacilli and is safe for chronic use.

Glycerol monolaurate (GML) is a fatty acid monoester that inhibits growth and exotoxin production of vaginal pathogens and cytokine production by vaginal epithelial cells. Because of these activities, and because of the importance of cytokine-mediated immune activation in human immunodeficiency virus type 1 (HIV-1) transmission to women, our laboratories are performing studies on the potential efficacy of GML as a topical microbicide to interfere with HIV-1 transmission in the simian immunodeficiency virus-rhesus macaque model. While GML is generally recognized as safe by the FDA for topical use, its safety for chronic use and effects on normal vaginal microflora in this animal model have not been evaluated. GML was therefore tested both in vitro for its effects on vaginal flora lactobacilli and in vivo as a 5% gel administered vaginally to monkeys. In vitro studies demonstrated that lactobacilli are not killed by GML; GML blocks the loss of their viability in stationary phase and does not interfere with lactic acid production. GML (5% gel) does not quantitatively alter monkey aerobic vaginal microflora compared to vehicle control gel. Lactobacilli and coagulase-negative staphylococci are the dominant vaginal aerobic microflora, with beta-hemolytic streptococci, Staphylococcus aureus, and yeasts sporadically present; gram-negative rods are not part of their vaginal flora. Colposcopy and biopsy studies indicate that GML does not alter normal mucosal integrity and does not induce inflammation; instead, GML reduces epithelial cell production of interleukin 8. The studies suggest that GML is safe for chronic use in monkeys when applied vaginally; it does not alter either mucosal microflora or integrity.

Rabbit Model for Superantigen-Mediated Lethal Pulmonary Disease.

Staphylococcus aureus is a highly significant cause of serious human infections in the USA. Many of these illnesses are mediated by interactions between the host immune system and staphylococcal superantigens (SAgs). Several of these severe staphylococcal infections are initiated in the lungs, making this an important site to study. Here, we describe the rabbit model for investigating the role of staphylococcal SAgs in pulmonary-associated lethal infection and intoxication.

Type A Francisella tularensis acid phosphatases contribute to pathogenesis.

Different Francisella spp. produce five or six predicted acid phosphatases (AcpA, AcpB, AcpC, AcpD, HapA and HapB). The genes encoding the histidine acid phosphatases (hapA, hapB) and acpD of F. tularensis subsp. Schu S4 strain are truncated or disrupted. However, deletion of HapA (FTT1064) in F. tularensis Schu S4 resulted in a 33% reduction in acid phosphatase activity and loss of the four functional acid phosphatases in F. tularensis Schu S4 (ΔABCH) resulted in a>99% reduction in acid phosphatase activity compared to the wild type strain. All single, double and triple mutants tested, demonstrated a moderate decrease in mouse virulence and survival and growth within human and murine phagocytes, whereas the ΔABCH mutant showed >3.5-fold decrease in intramacrophage survival and 100% attenuation of virulence in mouse. While the Schu S4 ΔABCH strain was attenuated in the mouse model, it showed only limited protection against wild type challenge. F. tularensis Schu S4 failed to stimulate reactive oxygen species production in phagocytes, whereas infection by the ΔABCH strain stimulated 5- and 56-fold increase in reactive oxygen species production in neutrophils and human monocyte-derived macrophages, respectively. The ΔABCH mutant but not the wild type strain strongly co-localized with p47 (phox) and replicated in macrophages isolated from p47 (phox) knockout mice. Thus, F. tularensis Schu S4 acid phosphatases, including the truncated HapA, play a major role in intramacrophage survival and virulence of this human pathogen.

An altered immune response, but not individual cationic antimicrobial peptides, is associated with the oral attenuation of Ara4N-deficient Salmonella enterica serovar Typhimurium in mice.

Salmonella enterica serovar Typhimurium (S. Typhimurium) uses two-component regulatory systems (TCRS) to respond to stimuli in the local microenvironment. Upon infection, the Salmonella TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals in the intestinal lumen and within host cells. TCRS-mediated gene expression results in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide resistance. The PmrA-regulated pmrHFIJKLM operon mediates 4-amino-4-deoxy-L-arabinose (Ara4N) production and attachment to the lipid A of LPS. A ΔpmrF S. Typhimurium strain cannot produce Ara4N, exhibits increased sensitivity to cationic antimicrobial peptide (CAMP)-mediated killing, and attenuated virulence in mice upon oral infection. CAMPs are predicted to play a role in elimination of Salmonella, and may activate PhoPQ and PmrAB in vivo, which could increase bacterial resistance to host defenses. Competition experiments between wild type (WT) and ΔpmrF mutant strains of S. Typhimurium indicated that selection against this mutant first occurs within the intestinal lumen early during infection. However, CRAMP and active cryptdins alone are not responsible for elimination of Ara4N-deficient bacteria in vivo. Investigation into the early immune response to ΔpmrF showed that it differed slightly from the early immune response to WT S. Typhimurium. Further investigation into the early immune response to infection of Peyer's patches suggests a role for IL-13 in the attenution of the ΔpmrF mutant strain. Thus, prominent CAMPs present in the mouse intestine are not responsible for the selection against the ΔpmrF strain in this location, but limited alterations in innate immune induction were observed that affect bacterial survival and virulence.

Cationic antimicrobial peptides serve as activation signals for the Salmonella Typhimurium PhoPQ and PmrAB regulons in vitro and in vivo.

Salmonella enterica serovar Typhimurium uses two-component regulatory systems (TCRSs) to respond to environmental stimuli. Upon infection, the TCRSs PhoP-PhoQ (PhoPQ) and PmrA-PmrB (PmrAB) are activated by environmental signals detected in the lumen of the intestine and within host cells. TCRS-mediated gene expression leads to upregulation of genes involved in lipopolysaccharide (LPS) modification and cationic antimicrobial peptide (CAMP) resistance. This research expands on previous studies which have shown that CAMPs can activate Salmonella TCRSs in vitro. The focus of this work was to determine if CAMPs can act as environmental signals for PhoPQ- and PmrAB-mediated gene expression in vitro, during infection of macrophages and in a mouse model of infection. Monitoring of PhoPQ and PmrAB activation using recombinase-based in vivo expression technology (RIVET), alkaline phosphtase and ß-galactosidase reporter fusion constructs demonstrated that S. Typhimurium PhoQ can sense CAMPs in vitro. In mouse macrophages, the cathelecidin CRAMP does not activate the PhoPQ regulon. Acidification of the Salmonella-containing vacuole activates PhoP- and PmrA-regulated loci but blocking acidification still does not reveal a role for CRAMP in TCRS activation in mouse macrophages. However, assays performed in susceptible wild type (WT), CRAMP knockout (KO), and matrilysin (a metalloproteinase necessary for activating murine α-defensins) KO mice suggest CRAMP, but not α-defensins, serve as a putative direct TCRS activation signal in the mouse intestine. These studies provide a better understanding of the in vivo environments that result in activation of these virulence-associated TCRSs.

Vaginal Staphylococcus aureus superantigen profile shift from 1980 and 1981 to 2003, 2004, and 2005.

We determined vaginal Staphylococcus aureus superantigens. Staphylococci were quantified from tampons/diaphragms in 2003 to 2005, with counts compared to those determined in 1980 and 1981. In 2003 to 2005, more women were colonized than in 1980 and 1981 (23 versus 12%). Enterotoxins G and I and enterotoxin-like superantigens M and N declined, but enterotoxin-like superantigens K, L, and Q increased.