Expression of virulence factors by Staphylococcus aureus grown in serum

Staphylococcus aureus produces many virulence factors, including toxins, immune-modulatory factors, and exoenzymes. Previous studies involving the analysis of virulence expression were mainly performed by in vitro experiments using bacterial medium. However, when S. aureus infects a host, the bacterial growth conditions are quite different from those in a medium, which may be related to the different expression of virulence factors in the host. In this study, we investigated the expression of virulence factors in S. aureus grown in calf serum. The expression of many virulence factors, including hemolysins, enterotoxins, proteases, and iron acquisition factors, was significantly increased compared with that in bacterial medium. In addition, the expression of RNA III, a global regulon for virulence expression, was significantly increased. This effect was partially restored by the addition of 300 μM FeCl₃ into serum, suggesting that iron depletion is associated with the increased expression of virulence factors in serum. In chemically defined medium without iron, a similar effect was observed. In a mutant with agr inactivated grown in serum, the expression of RNA III, psm, and sec4 was not increased, while other factors were still induced in the mutant, suggesting that another regulatory factor(s) is involved. In addition, we found that serum albumin is a major factor for the capture of free iron to prevent the supply of iron to bacteria grown in serum. These results indicate that S. aureus expresses virulence factors in adaptation to the host environment.     

Possible virulence factors of Staphylococcus aureus in a mouse septic model

Twenty clinical isolates of Staphylococcus aureus were examined to elucidate the virulence factors which are directly related to lethality in a mouse septic model. Heat or formalin treatment of the organism abolished the lethal activity of the live organism during challenge intravenously administered via the tail vein. Nevertheless, injection of ten times concentrated culture supernatant fluid (SUP) showed lethal activity in the mouse. However, there was no lethality when SUP was heated at 60 degrees C for 15 min. To examine variations of SUP lethality among strains, we collected 20 strains of S. aureus from four different hospitals. Then, we compared several factors for SUP lethality, which were the extracellular toxins and enzymes, such as toxic shock syndrome toxin 1, enterotoxin A, B, D, and hemolysins (alpha,beta,gamma), and also cytotoxic activity to human polymorphonuclear leukocytes and Vero cells. No difference was found among these factors except cytotoxic activity to Vero cells. Furthermore, we compared two strains in a mouse septic model according to the grade of bacteremia and lethal events. We found that mortality was higher with challenge by the strain whose SUP was lethal in comparison to the strain whose SUP was not lethal, even though the viable bacteria counts in the septic blood in both strains were not significantly different. This strongly supports the possibility that extracellular products, not the cell wall components, of S. aureus play the key role in the lethal event in this mouse septic model. In addition, among the extracellular products, those which have cytotoxic activity to Vero cells may contribute to the lethality in sepsis caused by S. aureus in this murine model.     

Evaluation of Staphylococcus aureus virulence factors using a silkworm model

Previous studies have indicated that the silkworm model is useful for identifying virulence genes of Staphylococcus aureus, a human pathogenic bacterium. Here we examined the scope of S.?aureus virulence factors that can be evaluated using the silkworm model. Gene-disrupted mutants of the agr locus, arlS gene and saeS gene, which regulate the expression of cell surface adhesins and hemolysins, exhibited attenuated virulence in silkworms. Mutants of the hla gene encoding α-hemolysin, the hlb gene encoding β-hemolysin, and the psmα and psmβ operons encoding cytolysins, however, showed virulence in silkworms indistinguishable from that of the parent strain. Thus, these S.?aureus cytolysins are not required for virulence in silkworms. In contrast, the gene-disrupted mutants of clfB, fnbB and sdrC, which encode cell-wall-anchored proteins, attenuated S.?aureus virulence in silkworms. In addition, the mutant of the srtA gene encoding sortase A, which anchors cell-wall proteins, showed attenuated virulence in silkworms. These findings suggest that the silkworm model can be used to evaluate S.?aureus cell-wall proteins and regulatory proteins as virulence factors.     

Iron depletion and virulence in Staphylococcus aureus

Abstract Staphylococcus aureus is able to grow in the presence of extremely low iron concentrations (0.04 μM). In iron-limiting conditions, this species develops alternative metabolic strategies such as highly efficient iron-uptake mechanisms which are only partially shared with S. epidermidis . Here we summarize the mechanisms induced by iron starvation in S. aureus in order to elucidate the virulence characteristics of this bacterium.     

Loss of virulence of revertants from Staphylococcus aureus L-phase variants in comparison with the parent strain

The L-forms of the Klecha 155 strain were induced in vitro by methicillin. After reversion, the strains were tested for 19 biochemical features and for susceptibility to penicillin, ampicillin and methicillin. Two reverted strains were less virulent for mice than the parent strain. It is concluded that the decrease in virulence of reverted Staph. aureus strains is correlated with changes in the chemical structure and composition of the cell wall after reversion from the L-phase variants.     

Sub-inhibitory membrane damage undermines Staphylococcus aureus virulence

We investigated antibacterial properties of a recently described membrane-active lipopeptide, C₁₀OOc₁₂O (decanoyl-ornithyl-ornithyl-dodecanoyl-ornithyl-amide) against Gram-positive bacteria (GPB). Minimal inhibitory concentrations (MICs) and kinetics were compared in culture media and plasma. Chemo-sensitization to antibiotics was determined using the checkerboard assay. Membrane damages were estimated using diverse membrane potential sensitive dyes. ATP levels and relevant enzymes activities were measured using commercial bioassay kits.While relatively weakly active in simple culture media, sub-MIC levels (~ten-fold) of C₁₀OOc₁₂O have significantly improved the antibacterial function of Human plasma. Mechanistic studies indicated that C₁₀OOc₁₂O-treated bacteria have sustained mild membrane damage(s) in association with rapid (within 2 min) but low (<10%) dissipation of the trans-membrane potential; Intracellular ATP levels were transiently reduced (~20%) whereas extracellular ATP increased only at MIC values; Sub-inhibitory concentrations were sufficient for inhibiting major agr-regulated virulence factors (lipase and α-toxin) and for sensitizing MRSA USA300 to the antibiotic oxacillin to the point of reverting the bacteria status from oxacillin-resistant to oxacillin-sensitive (i.e., oxacillin MIC was reduced from 32 to 0.1 mg/l).These findings argue that by means of mild depolarization, C₁₀OOc₁₂O affects the quorum sensing regulator in a manner that transiently weakens bacterial defenses, thereby enforcing studies that support the potential usefulness of fighting S. aureus (and possibly other GPB) infections, by targeting its virulence.     

Silkworm apolipophorin protein inhibits Staphylococcus aureus virulence

Silkworm hemolymph inhibits hemolysin production by Staphylococcus aureus. We purified a factor in the silkworm hemolymph responsible for this inhibitory activity. The final fraction with the greatest specific activity contained 220- and 74-kDa proteins. Determination of the N-terminal amino acid sequence revealed that the 220- and 74-kDa proteins were apolipophorin I and apolipophorin II, respectively, indicating that the factor was apolipophorin (ApoLp). The purified ApoLp fraction showed decreased expression of S. aureus hla encoding α-hemolysin, hlb encoding β-hemolysin, saeRS, and RNAIII, which activate the expression of these hemolysin genes. Injection of an anti-ApoLp antibody into the hemolymph increased the sensitivity of silkworms to the lethal effect of S. aureus. Hog gastric mucin, a mammalian homologue of ApoLp, decreased the expression of S. aureus hla and hlb. These findings suggest that ApoLp in the silkworm hemolymph inhibits S. aureus virulence and contributes to defense against S. aureus infection and that its activity is conserved in mammalian mucin.     

Wild-type variants of exopenicillinase from Staphylococcus aureus

1. Three variants of staphylococcal exopenicillinase (types A, B and C) can be distinguished on chemical, enzymological and immunological grounds. 2. Enzyme type A has a higher specific activity than that of type B, but has a similar combination affinity with anti-(exopenicillinase type A) serum. 3. Enzyme types A and C have a similar specific activity, but enzyme type C has a lower combination affinity for anti-(exopenicillinase type A) serum than has enzyme type A. 4. The sedimentation coefficients and amino acid analyses of the three enzyme types are similar. 5. All three enzyme types have small but significant differences in kinetics of action when hydrolysing benzylpenicillin, methicillin, cloxacillin and cephalosporin C. 6. Peptide maps, obtained from enzyme types A and C after digestion with trypsin, show that these two variants probably differ in the nature of only a very few amino acid residues. 7. Enzyme type B seems to be confined to staphylococci that are members of staphylococcal phage group II. Enzyme types A and C are produced by staphylococci that are members either of phage group I or III, but never group II. 8. The low specific enzyme activity and affinity of enzyme type B towards all penicillins tested suggest that this enzyme type has a lower `efficiency' in hydrolysing penicillin and therefore in protecting bacteria from the action of penicillin. This could account for the low incidence among `hospital staphylococci' of penicillin-resistant staphylococci that are members of phage group II.     

Metabolic control of virulence factor production in Staphylococcus aureus

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beta-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus

Antibiotics that interfere with DNA replication and cell viability activate the SOS response. In Staphylococcus aureus, the antibiotic-induced SOS response promotes replication and high-frequency horizontal transfer of pathogenicity island-encoded virulence factors. Here we report that β-lactams induce a bona fide SOS response in S. aureus, characterized by the activation of the RecA and LexA proteins, the two master regulators of the SOS response. Moreover, we show that β-lactams are capable of triggering staphylococcal prophage induction in S. aureus lysogens. Consequently, and as previously described for SOS induction by commonly used fluoroquinolone antibiotics, β-lactam-mediated phage induction also resulted in replication and high-frequency transfer of the staphylococcal pathogenicity islands, showing that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors.