Characterization of Ssc, an N-acetylgalactosamine-containing Staphylococcus aureus surface polysaccharide.

Whole genome sequencing has revealed that the genome of Staphylococcus aureus possesses an uncharacterized 5-gene operon (SAOUHSC_00088-00092 in strain 8325 genome) that encodes factors with functions related to polysaccharide biosynthesis and export, indicating the existence of a new extracellular polysaccharide species. We designate this locus as ssc for staphylococcal surface carbohydrate. We found that the ssc genes were weakly expressed and highly repressed by the global regulator MgrA. To characterize Ssc, Ssc was heterologously expressed in Escherichia coli and extracted by heat treatment. Ssc was also conjugated to AcrA from Campylobacter jejuni in E. coli using protein glycan coupling technology (PGCT). Analysis of the heat-extracted Ssc and the purified Ssc-AcrA glycoconjugate by tandem mass spectrometry revealed that Ssc is likely a polymer consisting of N-acetylgalactosamine. We further demonstrated that the expression of the ssc genes in S. aureus affected phage adsorption and susceptibility, suggesting that Ssc is surface-exposed. IMPORTANCE: Surface polysaccharides play crucial roles in the biology and virulence of bacterial pathogens. Staphylococcus aureus produces four major types of polysaccharides that have been well-characterized. In this study, we identified a new surface polysaccharide containing N-acetylgalactosamine (GalNAc). This marks the first report of GalNAc-containing polysaccharide in S. aureus. Our discovery lays the groundwork for further investigations into the chemical structure, surface location, and role in pathogenesis of this new polysaccharide.

Regulation of Staphylococcal Capsule by SarZ is SigA-Dependent.

Production of capsular polysaccharides in Staphylococcus aureus is transcriptionally regulated by a control region of the cap operon that consists of SigA- and SigB-dependent promoters. A large number of regulators have been shown to affect cap gene expression. However, regulation of capsule is only partially understood. Here we found that SarZ was another regulator that activated the cap genes through the SigA-dependent promoter. Gel electrophoresis mobility shift experiments revealed that SarZ is bound to a broad region of the cap promoter including the SigA-dependent promoter but mainly the downstream region. We demonstrated that activation of cap expression by SarZ was independent of MgrA, which also activated capsule through the SigA-dependent promoter. Our results further showed that oxidative stress with hydrogen peroxide (H2O2) treatments enhanced SarZ activation of cap expression, indicating that SarZ is able to sense oxidative stress to regulate capsule production. IMPORTANCE Expression of virulence genes in Staphylococcus aureus is affected by environmental cues and is regulated by a surprisingly large number of regulators. Much is still unknown about how virulence factors are regulated by environment cues at the molecular level. Capsule is an antiphagocytic virulence factor that is highly regulated. In this study, we found SarZ was an activator of capsule and that the regulation of capsule by SarZ was affected by oxidative stress. These results provide an example of how a virulence factor could be regulated in response to an environmental cue. As the host oxidative defense system plays an important role against S. aureus, this study contributes to a better understanding of virulence gene regulation and staphylococcal pathogenesis.

A Rat Model of Orthopedic Implant-Associated Infection for Identification of Staphylococcal Biofilm Proteins.

Secreted bacterial proteins are difficult to identify directly from an infection site due to a limited amount of bacteria and presence of a large quantity of host proteins. Here we describe a rat model of orthopedic implant that allows us to harvest bacterial biofilm materials sufficient for identification of bacterial proteins in the biofilm matrix by liquid chromatography-tandem MS (GeLC-MS/MS) analysis.

MgrA Activates Staphylococcal Capsule via SigA-Dependent Promoter.

Staphylococcus aureus capsule polysaccharide is an important antiphagocytic virulence factor. The cap genes are regulated at the promoter element (Pcap) upstream of the cap operon. Pcap, which consists of a dominant SigB-dependent promoter and a weaker upstream SigA-dependent promoter, is activated by global regulator MgrA. How MgrA activates capsule is unclear. Here, we showed that MgrA directly bound to the Pcap region and affected the SigA-dependent promoter. Interestingly, an electrophoretic mobility shift assay showed that MgrA bound to a large region of Pcap, mainly downstream of the SigA-dependent promoter. We further showed that the ArlRS two-component system and the Agr quorum sensing system activated capsule primarily through MgrA in the early growth phases.IMPORTANCE The virulence of Staphylococcus aureus depends on the expression of various virulence factors, which is governed by a complex regulatory network. We have been using capsule as a model virulence factor to study virulence gene regulation in S. aureus MgrA is one of the regulators of capsule and has a major effect on capsule production. However, how MgrA regulates capsule genes is not understood. In this study, we were able to define the mechanism involving MgrA regulation of capsule. In addition, we also delineated the role of MgrA in capsule regulatory pathways involving the key virulence regulators Agr and Arl. This study further advances our understanding of virulence gene regulation in S. aureus, an important human pathogen.

MgrA Negatively Impacts Staphylococcus aureus Invasion by Regulating Capsule and FnbA.

Virulence genes are regulated by a complex regulatory network in Staphylococcus aureus Some of the regulators are global in nature and affect many downstream genes. MgrA is a multiple-gene regulator that has been shown to activate genes involved in capsule biosynthesis and repress surface protein genes. The goal of this study was to demonstrate the biological significance of MgrA regulation of capsule and surface proteins. We found that strain Becker possessed one fibronectin-binding protein, FnbA, and that FnbA was the predominant protein involved in invasion of nonphagocytic HeLa cells. By genetic analysis of strains with different amounts of capsule, we demonstrated that capsule impeded invasion of HeLa cells by masking the bacterial cell wall-anchored protein FnbA. Using variants with different levels of mgrA transcription, we further demonstrated that MgrA negatively impacted invasion by activating the cap genes involved in capsule biosynthesis and repressing the fnbA gene. Thus, we conclude that MgrA negatively impacts cell invasion of S. aureus Becker by promoting capsule and repressing FnbA.

Contribution of hla Regulation by SaeR to Staphylococcus aureus USA300 Pathogenesis.

The SaeRS two-component system in Staphylococcus aureus is critical for regulation of many virulence genes, including hla, which encodes alpha-toxin. However, the impact of regulation of alpha-toxin by Sae on S. aureus pathogenesis has not been directly addressed. Here, we mutated the SaeR-binding sequences in the hla regulatory region and determined the contribution of this mutation to hla expression and pathogenesis in strain USA300 JE2. Western blot analyses revealed drastic reduction of alpha-toxin levels in the culture supernatants of SaeR-binding mutant in contrast to the marked alpha-toxin production in the wild type. The SaeR-binding mutation had no significant effect on alpha-toxin regulation by Agr, MgrA, and CcpA. In animal studies, we found that the SaeR-binding mutation did not contribute to USA300 JE2 pathogenesis using a rat infective endocarditis model. However, in a rat skin and soft tissue infection model, the abscesses on rats infected with the mutant were significantly smaller than the abscesses on those infected with the wild type but similar to the abscesses on those infected with a saeR mutant. These studies indicated that there is a direct effect of hla regulation by SaeR on pathogenesis but that the effect depends on the animal model used.

Repression of Capsule Production by XdrA and CodY in Staphylococcus aureus.

Capsule is one of many virulence factors produced by Staphylococcus aureus, and its expression is highly regulated. Here, we report the repression of capsule by direct interaction of XdrA and CodY with the capsule promoter region. We found, by footprinting analyses, that XdrA repressed capsule by binding to a broad region that extended from upstream of the -35 region of the promoter to the coding region of capA, the first gene of the 16-gene cap operon. Footprinting analyses also revealed that CodY bound to a large region that overlapped extensively with that of XdrA. We found that repression of the cap genes in the xdrA mutant could be achieved by the overexpression of codY but not vice versa, suggesting codY is epistatic to xdrA However, we found XdrA had no effect on CodY expression. These results suggest that XdrA plays a secondary role in capsule regulation by promoting CodY repression of the cap genes. Oxacillin slightly induced xdrA expression and reduced cap promoter activity, but the effect of oxacillin on capsule was not mediated through XdrA.IMPORTANCEStaphylococcus aureus employs a complex regulatory network to coordinate the expression of various virulence genes to achieve successful infections. How virulence genes are coordinately regulated is still poorly understood. We have been studying capsule regulation as a model system to explore regulatory networking in S. aureus In this study, we found that XdrA and CodY have broad binding sites that overlap extensively in the capsule promoter region. Our results also suggest that XdrA assists CodY in the repression of capsule. As capsule gene regulation by DNA-binding regulators has not been fully investigated, the results presented here fill an important knowledge gap, thereby further advancing our understanding of the global virulence regulatory network in S. aureus.

Proteomics of Staphylococcus aureus biofilm matrix in a rat model of orthopedic implant-associated infection.

The matrix proteins of Staphylococcus aureus biofilm have not been well defined. Previous efforts to identify these proteins were performed using in vitro systems. Here we use a proteomic approach to identify biofilm matrix proteins directly from infected bone implants using a rat model of orthopedic implant-associated S. aureus infection. Despite heavy presence of host proteins, a total of 28 and 105 S. aureus proteins were identified during acute infection and chronic infection, respectively. Our results show that biofilm matrix contains mostly intracellular cytoplasmic proteins and, to a much less extent, extracellular and cell surface-associated proteins. Significantly, leukocidins were identified in the biofilm matrix during chronic infection, suggesting S. aureus is actively attacking the host immune system even though they are protected within the biofilm. The presence of two surface-associated proteins, Ebh and SasF, in the infected bone tissue during acute infection was confirmed by immunohistochemistry. In addition, a large number of host proteins were found differentially expressed in response to S. aureus biofilm formed on bone implants.

RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus.

UNLABELLED: Staphylococcus aureus capsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of the cap operon. A 10-bp inverted repeat (IR) located 13 bp upstream of the -35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of the cap promoter. To search for potential proteins which directly interact with the cap promoter region (Pcap), we directly analyzed the proteins interacting with the Pcap DNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulate cap gene expression by specifically binding to the cap promoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed that rbsR was directly controlled by SigB and that RbsR was a repressor of the rbsUDK operon, involved in ribose uptake and phosphorylation. The repression of rbsUDK by RbsR could be derepressed by D-ribose. However, D-ribose did not affect RbsR activation of capsule. IMPORTANCE: Staphylococcus aureus is an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression of rbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits the rbs operon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence in S. aureus. Thus, this study further advances our understanding of staphylococcal virulence regulation.

SaeRS-dependent inhibition of biofilm formation in Staphylococcus aureus Newman.

The SaeRS two-component regulatory system of Staphylococcus aureus is known to affect the expression of many genes. The SaeS protein is the histidine kinase responsible for phosphorylation of the response regulator SaeR. In S. aureus Newman, the sae system is constitutively expressed due to a point mutation in saeS, relative to other S. aureus strains, which results in substitution of proline for leucine at amino acid 18. Strain Newman is unable to form a robust biofilm and we report here that the biofilm-deficient phenotype is due to the saeSP allele. Replacement of the Newman saeSP with saeSL, or deletion of saeRS, resulted in a biofilm-proficient phenotype. Newman culture supernatants were observed to inhibit biofilm formation by other S. aureus strains, but did not affect biofilm formation by S. epidermidis. Culture supernatants of Newman saeSL or Newman ΔsaeRS had no significant effect on biofilm formation. The inhibitory factor was inactivated by incubation with proteinase K, but survived heating, indicating that the inhibitory protein is heat-stable. The inhibitory protein was found to affect the attachment step in biofilm formation, but had no effect on preformed biofilms. Replacement of saeSL with saeSP in the biofilm-proficient S. aureus USA300 FPR3757 resulted in the loss of biofilm formation. Culture supernatants of USA300 FPR3757 saeSP, did not inhibit biofilm formation by other staphylococci, suggesting that the inhibitory factor is produced but not secreted in the mutant strain. A number of biochemical methods were utilized to isolate the inhibitory protein. Although a number of candidate proteins were identified, none were found to be the actual inhibitor. In an effort to reduce the number of potential inhibitory genes, RNA-Seq analyses were done with wild-type strain Newman and the saeSL and ΔsaeRS mutants. RNA-Seq results indicated that sae regulates many genes that may affect biofilm formation by Newman.

Trapping and identification of cellular substrates of the Staphylococcus aureus ClpC chaperone.

ClpC is an ATP-dependent Hsp100/Clp chaperone involved in protein quality control in low-GC Gram-positive bacteria. Previously, we found that ClpC affected the expression of a large number of genes, including capsule genes in Staphylococcus aureus. Here we constructed a His-tagged ClpC variant (ClpC(trap)) with mutations within the Walker B motifs to identify the direct substrates of ClpC by copurification with ClpC(trap) followed by gel electrophoresis combined with liquid chromatography-tandem mass spectrometry proteomics. We identified a total of 103 proteins that are potential substrates of ClpC in strain Newman. The direct protein-protein interaction of ClpC with a subset of the captured proteins was verified in a bacterial two-hybrid system. The captured proteins could be grouped into various functional categories, but most were related to proteins involved in the stress response. Several known ClpC substrates were captured, including ClpP, TrfA/MecA, ClpB, DnaK, DnaJ, GroL, RecA, and CodY, supporting the validity of our approach. Our results also revealed many new ClpC substrates, including AgrA, CcpA, RsbW, MurG, FtsA, SrtA, Rex, Atl, ClfA, and SbcC. Analysis of capsule production showed that three of the captured proteins, which were not previously known to be transcriptional regulators, did affect capsule production.

Activation of sarX by Rbf is required for biofilm formation and icaADBC expression in Staphylococcus aureus.

A major constituent of many Staphylococcus aureus biofilms is a polysaccharide known as the polysaccharide intercellular adhesin, or poly N-acetylglucosamine (PIA/PNAG). PIA/PNAG is synthesized by the 4 gene products of the icaADBC operon, which is negatively regulated by the divergently transcribed icaR gene. We previously reported the identification of a gene, rbf, involved in the positive transcriptional regulation of icaADBC transcription by repressing icaR in S. aureus strain 8325-4. However, we were unable to show binding of Rbf to DNA upstream of icaR or icaA, suggesting that Rbf may control expression of an unknown factor(s) that, in turn, regulates ica expression. Here we report that the unknown factor is SarX protein. Results from epistasis assays and genetic complementation analyses suggest that Rbf upregulates SarX, which then downregulates IcaR, thereby activating icaADBC. Electrophoretic mobility shift assays revealed that SarX protein bound to a sequence upstream of icaR within the icaA coding region. Cross-linking and immunoprecipitation experiments further suggested that Rbf binds to the sarX promoter in S. aureus. These results demonstrate that Rbf and SarX represent a regulatory cascade that promotes PIA-dependent biofilm formation in S. aureus.

Genetic regulation of the intercellular adhesion locus in staphylococci.

The formation of biofilms by Staphylococcus aureus and Staphylococcus epidermidis is an important aspect of many staphylococcal infections, most notably endocarditis, osteomyelitis and infections associated with indwelling medical devices. The major constituents of staphylococcal biofilms are polysaccharides, such as poly N-acetyl glucosamine (PIA/PNAG), cell surface and secreted bacterial proteins, and extracellular DNA. The exact composition of biofilms often varies considerably between different strains of staphylococci and between different sites of infection by the same strain. PIA/PNAG is synthesized by the products of four genes, icaADBC, that are encoded in a single operon. A fifth gene, icaR, is a negative regulator of icaADBC. Expression of icaADBC is tightly regulated, but can often be induced in vitro by growing staphylococci in the presence of high salt, high glucose, or ethanol. Regulation of icaADBC is complex and numerous regulatory factors have been implicated in control of icaADBC. Many of these are well known global transcriptional regulatory factors like SarA and sigmaB, whereas other regulators, such as IcaR, seem to affect expression of relatively few genes. Here, we will summarize how various regulatory factors affect the production of PIA/PNAG in staphylococci.

A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome.

BACKGROUND: Single-copy integration vectors based upon the site-specific recombination systems of bacteriophage are invaluable tools in the study of bacterial pathogenesis. The utility of such vectors is often limited, however, by the fact that integration often results in the inactivation of bacterial genes or has undesirable effects on gene transcription. The aim of this study is to develop an integration vector that does not have a detectable effect on gene transcription upon integration. FINDINGS: We have developed a single-copy integration system that enables the cloning vector to integrate at a specific engineered site, within an untranscribed intergenic region, in the chromosome of Staphylococcus aureus. This system is based on the lysogenic phage L54a site-specific recombination system in which the L54a phage (attP) and chromosome (attB) attachment sites, which share an 18-bp identical core sequence, were modified with identical mutations. The integration vector, pLL102, was constructed to contain the modified L54a attP site (attP2) that was altered at 5 nucleotide positions within the core sequence. In the recipient strain, the similarly modified attB site (attB2) was inserted in an intergenic region devoid of detectable transcription read-through. Integration of the vector, which is unable to replicate in S. aureus extrachromosomally, was achieved by providing the L54a integrase gene in a plasmid in the recipient. We showed that pLL102 integrated specifically at the engineered site rather than at the native L54a attB site and that integration did not have a significant effect on transcription of genes immediately upstream or downstream of the integration site. CONCLUSIONS: In this work, we describe an E. coli-S. aureus shuttle vector that can be used to introduce any cloned gene into the S. aureus chromosome at a select site without affecting gene expression. The vector should be useful for genetic manipulation of S. aureus and for marking strains for in vivo studies.

Rsp inhibits attachment and biofilm formation by repressing fnbA in Staphylococcus aureus MW2.

Biofilms contribute to virulence of Staphylococcus aureus. Formation of biofilms is multifactorial, involving polysaccharide, protein, and DNA components, which are controlled by various regulators. Here we report that deletion of the rsp gene resulted in an increase in biofilm formation in strain MW2, suggesting that Rsp is a repressor of biofilm formation. Using SDS-PAGE, we found that Rsp profoundly affected cell surface and secreted proteins. The rsp gene was transcribed monocistronically, and the transcripts were most abundant at the exponential growth phase. Microarray analyses revealed that Rsp represses 75 genes, including 9 genes encoding cell wall-anchored proteins, and activates 22 genes, including 5 genes encoding secreted proteases. Among these genes, fnbA, fnbB, sasG, and spa (which encode cell wall-anchored proteins) and splABCD (which encode secreted proteases) have been implicated in biofilm formation. To deconvolute Rsp's contribution to biofilm formation, we analyzed deletion mutants of these genes either in the wild-type or in the rsp mutant background. We found that fnbA deletion in the rsp mutant restored biofilm formation to the wild-type level, indicating that FnbA plays a major role in Rsp regulation of biofilm formation. Further studies revealed that Rsp inhibited biofilm formation at the stage of primary attachment through repressing fnbA. Rsp belongs to the AraC/XylS family of regulatory proteins. We expressed the putative Rsp DNA binding domain (RspDBD) in Escherichia coli and showed that RspDBD was able to specifically bind to a short DNA fragment containing the fnbA promoter, suggesting that Rsp represses fnbA expression by direct DNA binding.

Tricarboxylic acid cycle-dependent attenuation of Staphylococcus aureus in vivo virulence by selective inhibition of amino acid transport.

Staphylococci are the leading causes of endovascular infections worldwide. Commonly, these infections involve the formation of biofilms on the surface of biomaterials. Biofilms are a complex aggregation of bacteria commonly encapsulated by an adhesive exopolysaccharide matrix. In staphylococci, this exopolysaccharide matrix is composed of polysaccharide intercellular adhesin (PIA). PIA is synthesized when the tricarboxylic acid (TCA) cycle is repressed. The inverse correlation between PIA synthesis and TCA cycle activity led us to hypothesize that increasing TCA cycle activity would decrease PIA synthesis and biofilm formation and reduce virulence in a rabbit catheter-induced model of biofilm infection. TCA cycle activity can be induced by preventing staphylococci from exogenously acquiring a TCA cycle-derived amino acid necessary for growth. To determine if TCA cycle induction would decrease PIA synthesis in Staphylococcus aureus, the glutamine permease gene (glnP) was inactivated and TCA cycle activity, PIA accumulation, biofilm forming ability, and virulence in an experimental catheter-induced endovascular biofilm (endocarditis) model were determined. Inactivation of this major glutamine transporter increased TCA cycle activity, transiently decreased PIA synthesis, and significantly reduced in vivo virulence in the endocarditis model in terms of achievable bacterial densities in biofilm-associated cardiac vegetations, kidneys, and spleen. These data confirm the close linkage of TCA cycle activity and virulence factor production and establish that this metabolic linkage can be manipulated to alter infectious outcomes.

Rbf promotes biofilm formation by Staphylococcus aureus via repression of icaR, a negative regulator of icaADBC.

We previously reported the identification of a gene, rbf, involved in the regulation of biofilm formation by Staphylococcus aureus 8325-4. In an effort to study the mechanism of regulation, microarrays were used to compare the transcription profiles of the wild-type strain with an rbf mutant and an rbf overexpression strain of the clinical isolate UAMS-1. Among the genes affected by rbf overexpression are those of the intercellular adhesion (ica) locus; however, expression of these genes was not affected by an rbf deletion in the chromosome. The icaADBC genes are responsible for production of poly-N-acetylglucosamine (PNAG), a major constituent of biofilm. The icaR gene encodes a negative regulator of icaADBC. In UAMS-1 carrying an Rbf-encoding plasmid, Rbf was found to repress icaR transcription with a concomitant increase in icaADBC expression and increased PNAG and biofilm production relative to isogenic strains lacking the plasmid. Sequencing of the rbf gene from UAMS-1 showed that there was a 2-bp insertion affecting the 50th codon of the rbf open reading frame, suggesting that rbf is a pseudogene in UAMS-1. This finding explains why deletion of rbf had no effect on biofilm formation in UAMS-1. To further characterize the Rbf regulation on biofilm we compared biofilm formation, icaA and icaR transcription, and PNAG production in 8325-4 and its isogenic rbf and icaR single mutants and an rbf icaR double mutant. Our results are consistent with a model wherein rbf represses synthesis of icaR, which in turn results in derepression of icaADBC and increased PNAG production. Furthermore, purified rbf did not bind to the icaR or icaA promoter region, suggesting that rbf controls expression of an unknown factor(s) that represses icaR. The role of rbf in controlling the S. aureus biofilm phenotype was further demonstrated in a clinical strain, MW2.

Staphylococcus aureus Rbf activates biofilm formation in vitro and promotes virulence in a murine foreign body infection model.

We previously identified Rbf as an activator for biofilm formation on polystyrene surfaces in Staphylococcus aureus strain 8325-4. However, strain 8325-4 contains genetic mutations that may affect biofilm formation. To extend the observation to other strains, we used strain Newman, a weak biofilm producer, and strain UAMS-1, an osteomyelitis clinical strain, in this study. We found that mutations in the chromosomal rbf gene did not affect biofilm formation on polystyrene surfaces in these strains, but transformants of these strains carrying a multiple-copy plasmid containing the rbf gene formed stronger biofilms than the wild-type strains and the mutant strains. Using the flow cell method, we found that the chromosomal mutation in the rbf gene delayed biofilm formation, whereas strains with a plasmid containing the rbf gene accelerated biofilm formation in strains Newman and UAMS-1. These results led us to conclude that rbf is an activator of biofilm formation in different strains of S. aureus, although the degree of activation varies among strains. In a murine model of foreign body infection, the rbf mutations in strain Newman, but not in strain UAMS-1, reduced the bacterial survival rate in catheter lumen. However, UAMS-1 carrying multiple copies of rbf in a plasmid increased the bacterial survival rate. The animal studies therefore suggest that Rbf has a role in S. aureus virulence.

Regulation of cellular caveolin-1 protein expression in murine macrophages by microbial products.

Previously, we reported that expression of caveolin-1 in elicited peritoneal mouse macrophages was up-regulated by remarkably low (1.0-pg/ml) concentrations of Escherichia coli O111 lipopolysaccharide (LPS). Here we report that increases in caveolin-1 expression are manifested by different types of LPS, LPS-mimetic taxol, and heat-killed E. coli and to a much lesser extent by zymosan, polysaccharide-peptidoglycan, and heat-killed Staphylococcus aureus. Rhodobacter sphaeroides lipid A (RsDPLA) could not induce caveolin-1 expression in macrophages. Interestingly, polymyxin B (5 microg/ml) and RsDPLA show only a limited capacity to inhibit LPS-induced caveolin-1 expression. These findings suggest that expression of caveolin-1 in response to LPS may only partially be dependent upon lipid A. Recombinant tumor necrosis factor alpha marginally induces caveolin-1, suggesting that the ability of LPS to regulate caveolin-1 is not mediated primarily through an autocrine/paracrine mechanism involving this cytokine. Under conditions in which cellular levels of caveolin-1 are profoundly induced, no significant changes in TLR4 expression are observed. Of interest, caveolin-1 appears to localize to two cellular compartments, one associated with lipid rafts and a second associated with TLR4. Gamma interferon treatment inhibits the induction of caveolin-1 by LPS in macrophages. Inhibition of the p38 kinase-dependent pathway, but not the extracellular signal-regulated kinase pathway, effectively reduced the ability of LPS to mediate caveolin-1 up-regulation. Lactacystin, a potent inhibitor of the proteasome pathway, significantly modulates LPS-independent caveolin-1 expression, and lactacystin inhibits LPS-triggered caveolin-1 responses. These studies suggest that caveolin-1 up-regulation in response to LPS is likely to be proteasome dependent and triggered through the p38 kinase pathway.