Mei G. Lei

Differential Expression of Caveolin-1 in Lipopolysaccharide-Activated Murine Macrophages

ABSTRACT Five reciprocal cycles of subtractive hybridization using cDNA generated from fibroblasts with normal lipopolysaccharide (LPS) responsiveness (lpsn) and from hyporesponsive (lpsd) fibroblasts have led to the finding that caveolin-1 is expressed at markedly higher levels of mRNA inlpsd than in lpsnfibroblasts. Caveolin-1 message can also be readily detected via reverse transcription-PCR in the RAW264.7 and J774.1 macrophage-like cell lines as well as in primary thioglycolate (TG)-elicited mouse peritoneal macrophages. In RAW264.7 cells, both caveolin-1 mRNA and protein levels are down-regulated by LPS. In TG-elicited C3HeB/FeJ peritoneal macrophages, in contrast, expression of both caveolin-1 protein and mRNA is up-regulated in vitro in response to LPS stimulation. The up-regulation of caveolin-1 protein expression in C3HeB/FeJ peritoneal macrophages can be demonstrated at concentrations as low as 1.0 pg of LPS/ml. However, LPS concentrations approximately 4 orders of magnitude higher (104 pg/ml) were required to stimulate the LPS-hyporesponsive C3H/HeJ mice peritoneal macrophages such that significant caveolin-1 protein up-regulation was detected. Caveolin-1, a principal component of plasmalemmal caveolae, has been reported as a potentially important regulator for signal transduction during cellular stimulation. The results described in this report suggest that caveolin-1 expression may be associated with LPS signaling/internalization.

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.

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

ABSTRACT 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.

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 Rsps 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.

Regulation of Cellular Caveolin-1 Protein Expression in Murine Macrophages by Microbial Products

ABSTRACT 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 μg/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.

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.

Staphylococcus aureus Rbf Activates Biofilm Formation In Vitro and Promotes Virulence in a Murine Foreign Body Infection Model

ABSTRACT 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.

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.

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.