Examination of the Staphylococcus aureus nitric oxide reductase (saNOR) reveals its contribution to modulating intracellular NO levels and cellular respiration.

Staphylococcus aureus nitrosative stress resistance is due in part to flavohemoprotein (Hmp). Although hmp is present in all sequenced S. aureus genomes, 37% of analyzed strains also contain nor, encoding a predicted quinol-type nitric oxide (NO) reductase (saNOR). DAF-FM staining of NO-challenged wild-type, nor, hmp and nor hmp mutant biofilms suggested that Hmp may have a greater contribution to intracellular NO detoxification relative to saNOR. However, saNOR still had a significant impact on intracellular NO levels and complemented NO detoxification in a nor hmp mutant. When grown as NO-challenged static (low-oxygen) cultures, hmp and nor hmp mutants both experienced a delay in growth initiation, whereas the nor mutant's ability to initiate growth was comparable with the wild-type strain. However, saNOR contributed to cell respiration in this assay once growth had resumed, as determined by membrane potential and respiratory activity assays. Expression of nor was upregulated during low-oxygen growth and dependent on SrrAB, a two-component system that regulates expression of respiration and nitrosative stress resistance genes. High-level nor promoter activity was also detectable in a cell subpopulation near the biofilm substratum. These results suggest that saNOR contributes to NO-dependent respiration during nitrosative stress, possibly conferring an advantage to nor+ strains in vivo.

Arginine deiminase in Staphylococcus epidermidis functions to augment biofilm maturation through pH homeostasis.

Allelic replacement mutants were constructed within arginine deiminase (arcA1 and arcA2) to assess the function of the arginine deiminase (ADI) pathway in organic acid resistance and biofilm formation of Staphylococcus epidermidis 1457. A growth-dependent acidification assay (pH ∼5.0 to ∼5.2) determined that strain 1457 devoid of arginine deiminase activity (1457 ΔADI) was significantly less viable than the wild type following depletion of glucose and in the presence of arginine. However, no difference in viability was noted for individual 1457 ΔarcA1 (native) or ΔarcA2 (arginine catabolic mobile element [ACME]-derived) mutants, suggesting that the native and ACME-derived ADIs are compensatory in S. epidermidis. Furthermore, flow cytometry and electron paramagnetic resonance spectroscopy results suggested that organic acid stress resulted in oxidative stress that could be partially rescued by the iron chelator dipyridyl. Collectively, these results suggest that formation of hydroxyl radicals is partially responsible for cell death via organic acid stress and that ADI-derived ammonia functions to counteract this acid stress. Finally, static biofilm assays determined that viability, ammonia synthesis, and pH were reduced in strain 1457 ΔADI following 120 h of growth in comparison to strain 1457 and the arcA1 and arcA2 single mutants. It is hypothesized that ammonia synthesis via the ADI pathway is important to reduce pH stress in specific microniches that contain high concentrations of organic acids.

Fatty acids and monoacylglycerols inhibit growth of Staphylococcus aureus.

Staphylococcus aureus causes a variety of human infections including toxic shock syndrome, osteomyelitis, and mastitis. Mastitis is a common disease in the dairy cow, and S. aureus has been found to be a major infectious organism causing mastitis. The objectives of this research were to determine which FA and esterified forms of FA were inhibitory to growth of S. aureus bacteria. FA as well as their mono-, di-, and triacylglycerol forms were tested for their ability to inhibit a human toxic shock syndrome clinical isolate (MN8) and two S. aureus clinical bovine mastitis isolates (305 and Novel). The seven most potent inhibitors across all strains tested by minimum inhibitory concentration analysis included lauric acid, glycerol monolaurate, capric acid, myristic acid, linoleic acid, cis-9, trans-11 conjugated linoleic acid, and trans-10, cis-12 conjugated linoleic acid. Some of these lipids were chosen for 48-h growth curve analysis with a bovine mastitis S. aureus isolate (Novel) at doses of 0, 20, 50, and 100 microg/mL except myristic acid, which was tested at 0, 50, 100, and 200 microg/mL. The saturated FA (lauric, capric, myristic) and glycerol monolaurate behaved similarly and reduced overall growth. In contrast, the polyunsaturated FA (linoleic and cis-9, trans-11 conjugated linoleic acid) delayed the time to initiation of exponential growth in a dose-dependent fashion. The results suggest that lipids may be important in the control of S. aureus during an infection.

Bovine mammary immune response to an experimental intramammary infection with a Staphylococcus aureus strain containing a gene for staphylococcal enterotoxin C1.

Staphylococcal enterotoxin C (SEC), a superantigen, is the most frequently expressed enterotoxin by bovine strains of Staphylococcus aureus causing mastitis. To examine the possible impact of SEC on the immune response of the bovine mammary gland, we monitored changes in lymphocyte subpopulations in mammary glands of four lactating cows after intramammary instillation of S. aureus strain Rn4220 transformed with a plasmid containing a gene coding for SEC1. Four other lactating cows received the same strain transformed with the plasmid without the SEC1 gene (positive control), and four cows were untreated (negative control). Mammary quarter milk samples for somatic cell count (SCC) analysis and determination of N-acetyl-beta-D-glucosimindase (NAGase) activity levels were collected daily for 21 d postinstillation. Flow cytometry utilizing three-color analysis was used to phenotype lymphocyte subpopulations isolated from milk samples collected on d 0, 4, 7, 11, 14, 18, and 21 postinstillation from all the cows. Milk from mammary gland halves (positive control and experimental) or all mammary quarters (negative control) was collected for flow cytometric analysis. Increased NAGase activity, SCC, and isolated S. aureus demonstrated that infection was established in mammary quarters intrammarily instilled with bacteria. There were no significant differences (P > 0.05) in the proportions of BoCD4 helper T lymphocytes or BoCD8 cytotoxic T lymphocytes between the two infected treatment groups. There was a significant day x treatment difference of the proportion of a gammadelta T cell subpopulation that did not express BoCD2, but did express the ACT2 activation molecule and a significant treatment difference of a gammadelta T cell subpopulation that expressed BoCD2, but not the ACT2 activation molecule (P < 0.05). Results do not support the hypothesis that the presence of the gene for SEC1 alters the mammary BoCD4 or BoCD8 T lymphocyte response to infection.

Molecular characterization of a novel Staphylococcus aureus serine protease operon.

The present study identified and characterized a unique operon (spl) encoding six serine protease-like proteins. In addition, native Spl proteins were isolated and characterized. Typical of most exoproteins, the spl gene products contain putative 35- or 36-amino-acid signal peptides. The Spl proteins share 44 to 95% amino acid sequence identity with each other and 33 to 36% sequence identity with V8 protease. They also contain amino acids found in catalytic triads of enzymes in the trypsin-like serine protease family, and SplB and SplC were shown to degrade casein. The spl operon is transcribed on a 5.5-kb transcript, but several nonrandom degradation products of this transcript were also identified. Similar to other S. aureus exoprotein genes, the spl operon is maximally expressed during the transition into stationary phase and is positively controlled by the Agr virulence factor regulator. The Sar regulatory system did not affect spl operon expression. PCR analysis revealed the presence of the spl operon in 64% of the S. aureus isolates tested, although one spl operon-negative isolate was shown to contain at least two of the spl genes. Finally, intraperitoneal injection of an spl operon deletion mutant revealed no major differences in virulence compared to the parental strain.

Staphylococcal fibronectin binding protein interacts with heat shock protein 60 and integrins: role in internalization by epithelial cells.

We reported previously that internalization of Staphylococcus aureus by nonprofessional phagocytes involves an interaction between fibronectin (Fn) binding protein (FnBP) and the host cell, resulting in signal transduction, tyrosine kinase activity, and cytoskeletal rearrangement (K. Dziewanowska, J. M. Patti, C. F. Deobald, K. W. Bayles, W. R. Trumble, and G. A. Bohach, Infect. Immun. 67:4673-4678, 1999). The goal of the present study was to identify the host molecules responsible for uptake of the organism through an interaction with FnBP. First, Fn was required for internalization. Addition of small amounts of exogenous Fn stimulated the uptake of S. aureus by HEp-2 cells, which are deficient in Fn synthesis. Fn antibodies blocked internalization of the organism by MAC-T cell monolayers, a bovine epithelial cell line which expresses Fn. Second, a monoclonal antibody (MAb) specific for beta(1) integrins dramatically reduced S. aureus invasion, suggesting that the formation of a Fn bridge linking the host cell beta(1) integrin and FnBP precedes internalization. However, ligand blotting of cell membrane proteins with a functional fragment of FnBP consistently identified an additional approximately 55-kDa receptor on both human and bovine epithelial cells. This protein was purified and identified by N-terminal microsequencing as heat shock protein 60 (Hsp60). The interaction between FnBP and Hsp60 also occurred when the whole cells were used. Cell membrane localization of Hsp60 was confirmed by biotinylation with an agent nonpermeable to the cell membrane. Pretreatment of epithelial cells with a MAb specific for eukaryotic Hsp60 significantly reduced internalization of S. aureus. Combined, these results suggest that the FnBP binds directly to both Hsp60 and Fn and is linked to beta(1) integrins through a Fn bridge. The simultaneous involvement of Fn and two host cell ligands, beta(1) integrins and Hsp60, suggests that FnBP is a multifunctional adhesin that mediates internalization in a manner similar to that proposed for OpaA, the Neisseria gonorrhoeae FnBP homolog (J. P. M. van Putten, T. D. Duensing, and R. L. Cole, Mol. Microbiol. 29:369-379, 1998).

Analysis of genetic elements controlling Staphylococcus aureus lrgAB expression: potential role of DNA topology in SarA regulation.

Penicillin-induced killing and murein hydrolase activity in Staphylococcus aureus are dependent on a variety of regulatory elements, including the LytSR two-component regulatory system and the virulence factor regulators Agr and Sar. The LytSR effects on these processes can be explained, in part, by the recent finding that a LytSR-regulated operon, designated lrgAB, affects murein hydrolase activity and penicillin tolerance. To examine the regulation of lrgAB expression in greater detail, we performed Northern blot and promoter fusion analyses. Both methods revealed that Agr and Sar, like LytSR, positively regulate lrgAB expression. A mutation in the agr locus reduced lrgAB expression approximately sixfold, while the sar mutation reduced lrgAB expression to undetectable levels. cis-acting regulatory elements involved in lrgAB expression were identified by fusing various fragments of the lrgAB promoter region to the xylE reporter gene and integrating these constructs into the chromosome. Catechol 2,3-dioxygenase assays identified DNA sequences, including an inverted repeat and intrinsic bend sites, that contribute to maximal lrgAB expression. Confirmation of the importance of the inverted repeat was achieved by demonstrating that multiple copies of the inverted repeat reduced lrgAB promoter activity, presumably by titrating out a positive regulatory factor. The results of this study demonstrate that lrgAB expression responds to a variety of positive regulatory factors and suggest that specific DNA topology requirements are important for optimal expression.

The bactericidal action of penicillin: new clues to an unsolved mystery.

The Staphylococcus aureus lrgAB operon was recently shown to inhibit extracellular murein hydrolase activity and increase tolerance to penicillin. Further characterization of this operon could provide novel insight into the dynamics of S. aureus cell wall metabolism and the mechanism of penicillin-induced lethality.

Apoptosis induced by Staphylococcus aureus in epithelial cells utilizes a mechanism involving caspases 8 and 3.

In this study, we demonstrate that the mechanism by which Staphylococcus aureus induces apoptosis in bovine mammary epithelial (MAC-T) cells involves caspases 8 and 3, two key components of a proteolytic cascade leading to apoptosis. In addition, internalized S. aureus induces expression of the inflammatory cytokines tumor necrosis factor alpha and interleukin-1beta by MAC-T cells. These data suggest that the internalization of S. aureus could induce specific cellular responses in vivo that may ultimately impact the course of infection.

The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance.

Previous studies in our laboratory have shown that the Staphylococcus aureus LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a lrgAB mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the lrgAB mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the lrgAB defect by providing the lrgAB genes in trans restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the lrgAB mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the lrgAB mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the lrgAB operon was shown to be maximally expressed. This effect of the lrgAB mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the lrgAB mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which lrgAB expression was shown to be minimal. However, expression of the lrgAB operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of S. aureus involves a novel regulator of murein hydrolase activity.

Fibronectin binding protein and host cell tyrosine kinase are required for internalization of Staphylococcus aureus by epithelial cells.

Staphylococcus aureus expresses several surface proteins that promote adherence to host cell extracellular matrix proteins, including fibronectin (Fn). Since this organism has recently been shown to be internalized by nonprofessional phagocytes, a process that typically requires high-affinity binding to host cell receptors, we investigated the role of its Fn binding proteins (FnBPs) and other surface proteins in internalization by the bovine mammary gland epithelial cell line (MAC-T). Efficient internalization of S. aureus 8325-4 required expression of FnBPs; an isogenic mutant (DU5883), not expressing FnBPs, was reduced by more than 95% in its ability to invade MAC-T cells. Moreover, D3, a synthetic peptide derived from the ligand binding domain of FnBP, inhibited the internalization of the 8325-4 strain in a dose-dependent fashion and the efficiency of staphylococcal internalization was partially correlated with Fn binding ability. Interestingly, Fn also inhibited the internalization and adherence of S. aureus 8325-4 in a dose-dependent manner. In contrast to internalization, adherence of DU5883 to MAC-T was reduced by only approximately 40%, suggesting that surface binding proteins, other than FnBPs, can mediate bacterial adherence to cells. Adherence via these proteins, however, does not necessarily result in internalization of the staphylococci. An inhibitor of protein tyrosine kinase, genistein, reduced MAC-T internalization of S. aureus by 95%, indicating a requirement for a host signal transduction system in this process. Taken together, these results indicate that S. aureus invades nonprofessional phagocytes by a mechanism requiring interaction between FnBP and the host cell, leading to signal transduction and subsequent rearrangement of the host cell cytoskeleton.

Staphylococcus aureus Agr and Sar global regulators influence internalization and induction of apoptosis.

Staphylococcus aureus was recently shown to be internalized by and to induce apoptosis in a bovine mammary epithelial cell line, suggesting that these processes could be involved in staphylococcal pathogenesis or persistence. To examine the role of virulence factor regulators during internalization, mutant agr and sar strains of S. aureus were analyzed for their abilities to enter and induce apoptosis in epithelial cells. Like a previously characterized bovine mastitis isolate, the standard laboratory strain, RN6390 (wild type), entered the epithelial cells and subsequently induced apoptosis. In contrast, the mutant strains RN6911 (agr), ALC136 (sar), and ALC135 (agr sar) were internalized by the cultured cells at levels reproducibly greater than that for RN6390 but failed to induce apoptosis. The internalization of S. aureus was affected by growth phase, suggesting a role for agr-regulated surface proteins in this process. Furthermore, the ability to induce apoptosis required metabolically active intracellular bacteria. These data indicate that the ability of S. aureus to enter mammalian cells and induce apoptosis is dependent on factors regulated by Agr and Sar. Since transcriptional control by these global regulators is mediated by quorum-sensing and environmental factors, staphylococci may have the potential to induce several alternative effects on cells from an intracellular environment. A model for the function of the agr locus in the context of internalization, intracellular persistence, and dissemination is proposed.

Opposing roles of the Staphylococcus aureus virulence regulators, Agr and Sar, in Triton X-100- and penicillin-induced autolysis.

The regulation of murein hydrolases is a critical aspect of peptidoglycan growth and metabolism. In the present study, we demonstrate that mutations within the Staphylococcus aureus virulence factor regulatory genes, agr and sar, affect autolysis, resulting in decreased and increased autolysis rates, respectively. Zymographic analyses of these mutant strains suggest that agr and sar exert their effects on autolysis, in part, by modulating murein hydrolase expression and/or activity.

Intracellular Staphylococcus aureus escapes the endosome and induces apoptosis in epithelial cells.

We examined the invasion of an established bovine mammary epithelial cell line (MAC-T) by a Staphylococcus aureus mastitis isolate to study the potential role of intracellular survival in the persistence of staphylococcal infections. S. aureus cells displayed dose-dependent invasion of MAC-T cells and intracellular survival. An electron microscopic examination of infected cells indicated that the bacteria induced internalization via a mechanism involving membrane pseudopod formation and then escaped into the cytoplasm following lysis of the endosomal membrane. Two hours after the internalization of S. aureus, MAC-T cells exhibited detachment from the matrix, rounding, a mottled cell membrane, and vacuolization of the cytoplasm, all of which are indicative of cells undergoing programmed cell death (apoptosis). By 18 h, the majority of the MAC-T cell population exhibited an apoptotic morphology. Other evidence for apoptosis was the generation of MAC-T cell DNA fragments differing in size by increments of approximately 180 bp and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling of the fragmented nuclear DNA of the infected host cells. These results demonstrate that after internalization S. aureus escapes the endosome and induces apoptosis in nonprofessional phagocytes.

Transcription analysis of the Staphylococcus aureus gene encoding penicillin-binding protein 4.

The high level of cross-linking found in Staphylococcus aureus peptidoglycan is dependent on the low-molecular-weight penicillin-binding protein PBP4. Recently, the PBP4 gene, pbpD, was cloned and shown to be adjacent to and divergently transcribed relative to the putative ABC-type transporter gene, abcA. Disruption of abcA (in strain KB400) was previously shown to result in heightened resistance to several antibiotics known to interact with PBP4, suggesting that the regulation of pbpD is affected by abcA. In this report, this hypothesis was confirmed by use of a Northern (RNA) blot analysis which revealed increased accumulation of pbpD-specific transcripts in KB400 compared to that in the wild-type strain, 8325-4. By using reverse-phase high-performance liquid chromatography to examine the structure of the peptidoglycan, it was demonstrated that the increased expression of pbpD resulted in an increased level of peptidoglycan cross-linking in the staphylococcal cell wall. Promoter fusion studies demonstrated that the abcA mutation caused approximately 7-fold and 100-fold increases in pbpD and abcA promoter activities, respectively. Primer extension experiments revealed that these genes have long, untranslated leader sequences that result in a transcriptional overlap of 80 bp. Interestingly, deletion of a 26-bp region containing an inverted repeat sequence resulted in the loss of expression from both the abcA and the pbpD promoters. These data provide evidence that abcA and pbpD are under the control of a common regulatory mechanism that may involve the transport function of the abcA gene product.

Identification of LytSR-regulated genes from Staphylococcus aureus.

In this report, the characterization of a Staphylococcus aureus operon containing two LytSR-regulated genes, lrgA and lrgB, is described. Sequence and mutagenesis studies of these genes suggest that lrgA encodes a murein hydrolase exporter similar to bacteriophage holin proteins while lrgB may encode a protein having murein hydrolase activity.

Identification and characterization of the pckA gene from Staphylococcus aureus.

The Staphylococcus aureus pckA gene was identified and characterized. A pckA mutant lacked detectable phosphoenolpyruvate carboxykinase activity and grew poorly in the absence of glucose. Both enzymatic activity and pckA promoter activity in wild-type cells grown in the absence of glucose were at least 22-fold greater than activities in cells grown in the presence of glucose.

Identification and molecular characterization of a putative regulatory locus that affects autolysis in Staphylococcus aureus.

Previously in our laboratory, a PCR-based strategy was used to isolate potential sensor gene fragments from the Staphyloccus aureus genome. One DNA fragment was isolated that shared strong sequence similarity to genes encoding bacterial sensor proteins, indicating that it originated from within a potential staphylococcal sensor protein gene. In this study, the DNA surrounding the PCR product origin was cloned and sequenced. This analysis revealed the presence of two genes, termed lytS and lytR, whose deduced amino acid sequences were similar to those of members of the two-component regulatory system family of proteins. S. aureus cells containing an insertional disruption of lytS exhibited a marked propensity to form aggregates in liquid culture, suggesting that alterations in cell surface components exist in this strain. Transmission electron microscopic examination of these cells revealed that the cell surface was rough and diffuse and that a large proportion of the cell population had lysed. The lytS mutant also exhibited increased autolysis and an altered level of murein hydrolase activity produced compared with the parental strain, NCTC 8325-4. These data suggest that the lytS and lytR gene products control the rate of autolysis in S. aureus by affecting the intrinsic murein hydrolase activity associated with the cell.

Analysis of Staphylococcus aureus genes encoding penicillin-binding protein 4 and an ABC-type transporter.

A region of the Staphylococcus aureus chromosome has been isolated that contains the gene encoding penicillin-binding protein 4 (PBP4), as well as abcA, a gene that encodes a protein with strong sequence similarity to the ABC transporter family of proteins. A disruption in abcA by Campbell-type integration results in cells that display an increased resistance to methicillin and cefoxitin, two antibiotics known to interact with low-molecular-weight PBPs. Based on these observations, a potential regulatory link between these two genes is discussed.

A genetic and molecular characterization of the recA gene from Staphylococcus aureus.

Previous studies have identified mutant strains of Staphylococcus aureus that have deficiencies in genetic recombination and DNA repair. Although these phenotypes were tentatively attributed to mutations within the S. aureus recA gene, experimental evidence to confirm this has never been reported. To characterize recA from S. aureus, we first isolated transposon insertion mutations that were in close proximity to the recA-like mutation (uvs-568) in strain 112 UVS-1. This allowed for the mobilization of the uvs-568 mutation into strain RN4220, the common laboratory strain of S. aureus. Next, using Bacillus subtilis recA as a probe, we cloned S. aureus recA and determined its nucleotide sequence. The deduced amino acid (aa) sequence of RecA contained 347 aa and was 74% identical to B. subtilis RecA. Using a cloned DNA fragment originating from within S. aureus recA, we then constructed a recA null mutant strain, designated KB103, which exhibited the same phenotypic characteristics imposed by the uvs-568 mutation in the same background. Furthermore, genetic and physical mapping of S. aureus recA placed it in the same region as the uvs-568 mutation. These data strongly suggest that these mutations represent different alleles of the same recA gene.