Implications of autophagy in anthrax pathogenicity.

The etiological agent for anthrax is Bacillus anthracis, which produces lethal toxin (LT) that exerts a myriad of effects on many immune cells. In our previous study, it was demonstrated that LT and protective antigen (PA) induce autophagy in mammalian cells. Preliminary results suggest that autophagy may function as a cellular defense mechanism against LT-mediated toxemia. This degradation pathway may also be relevant to other aspects of the immune response in both innate and adaptive immunity. Understanding the role of autophagy in response to anthrax infection and the possibility of modulating this degradation pathway as potential countermeasures are subjects for further investigation.

Induction of autophagy by anthrax lethal toxin.

Autophagy is an evolutionary conserved intracellular process whereby cells break down long-lived proteins and organelles. Accumulating evidences suggest increasing physiological significance of autophagy in pathogenesis of infectious diseases. Anthrax lethal toxin (LT) exerts its influence on numerous cells and herein, we report a novel effect of LT-induced autophagy on mammalian cells. Several autophagy biochemical markers including LC3-II conversion, increased punctuate distribution of GFP-LC3 and development of acidic vesicular organelles (AVO) were detected in cells treated with LT. Analysis of individual LT component revealed a moderate increase in LC3-II conversion for protective antigen-treated cells, whereas the LC3-II level in lethal factor-treated cells remained unchanged. In addition, our preliminary findings suggest a protective role of autophagy in LT intoxication as autophagy inhibition resulted in accelerated cell death. This study presents a hitherto undescribed effect of LT-induced autophagy on cells and provides the groundwork for future studies on the implication of autophagy in anthrax pathogenesis.

Diversity of escape variant mutations in Simian virus 40 large tumor antigen (SV40 Tag) epitopes selected by cytotoxic T lymphocyte (CTL) clones.

To better understand the relationship between epitope variation and tumor escape from immune surveillance, SV40 T antigen-transformed B6/K-0 cells were subjected to selection with individual CTL clones specific for the SV40 T antigen H-2D(b)-restricted epitopes I or V. CTL-resistant populations were isolated from a majority of the selection cultures and substituted epitope sequences were identified within most of the resistant populations. Tag sequences deleted of all or portions of the selection-targeted epitope were identified, but in lower numbers compared to epitope sequences bearing single residue substitutions. Relatively few flanking residue substitutions were identified, and only in epitope I-targeted selections. The diversity (numbers and epitope residue locations) of substituted epitope residue positions varied between selections. These findings suggest that the scope of spontaneously occurring mutations that could allow for escape from individual CD8+ T cell clones is large.

Lysostaphin-resistant variants of Staphylococcus aureus demonstrate reduced fitness in vitro and in vivo.

Lysostaphin is under development as a therapy for serious staphylococcal infections. During preclinical development, lysostaphin-resistant Staphylococcus aureus variants have occasionally been reported in vitro and in vivo. The acquisition of resistance to this drug, however, leads to a significant increase in beta-lactam antibiotic susceptibility, rendering methicillin-resistant S. aureus (MRSA) strains functionally methicillin susceptible. In this study, we have demonstrated that the development of lysostaphin resistance by two strains of MRSA also led to a loss of fitness in the variants. Consistent with the mutations found in previously reported lysostaphin-resistant S. aureus variants, these two variants had mutations in their femA genes, resulting in nonfunctional FemA proteins and, thus, monoglycine cross bridges in the peptidoglycan. The diminished fitness of the lysostaphin-resistant variants was reflected by (i) a reduced logarithmic growth rate, with the variants being outcompeted in cocultures by their wild-type parental strains; (ii) increased susceptibility to elevated temperatures; and (iii) at least fivefold less virulence of the lysostaphin-resistant variants than their wild-type strains in a mouse kidney infection model, with the lysostaphin-resistant variants being outcompeted in coinfections with their wild-type parental strains. During a 14-day serial passage without selective pressure, the lysostaphin-resistant variants failed to develop compensatory mutations which restored their fitness. These results suggest that should lysostaphin resistance due to an alteration in the FemA function emerge in S. aureus during therapy with lysostaphin, the resistant variants would be less fit and less virulent, and, in addition, infections with these strains would be easily treatable with beta-lactam antibiotics.

Comparison of four methods for determining lysostaphin susceptibility of various strains of Staphylococcus aureus.

Lysostaphin is an endopeptidase that cleaves the pentaglycine cross-bridges of the staphylococcal cell wall rapidly lysing the bacteria. Recently, lysostaphin has been examined for its potential to treat infections and to clear Staphylococcus aureus nasal colonization, requiring a reliable method for determining the lysostaphin susceptibility of strains of S. aureus. We compared four methods for determining the lysostaphin susceptibility of 57 strains of methicillin-sensitive S. aureus, methicillin-resistant S. aureus, vancomycin intermediately susceptible S. aureus (VISA), mupirocin-resistant S. aureus, and various defined genetic mutants of S. aureus. Three reference lysostaphin-resistant S. aureus variants were also included in the assays as negative controls. The assays examined included turbidity, MIC, minimum bactericidal concentration (MBC), and disk diffusion assays. All of the strains of S. aureus tested, including a VISA strain which had previously been reported to be lysostaphin resistant, were susceptible to lysostaphin by all four methods. The three reference lysostaphin-resistant variants were resistant by all four methods. The disk diffusion assay was the simplest method to differentiate lysostaphin-susceptible S. aureus strains from lysostaphin-resistant variants, while the MBC assay could be used as a follow-up assay if required. In the disk diffusion assay, all strains of S. aureus tested revealed zones of inhibition of >/=11 mm using a 50-microg lysostaphin disk, while the three reference lysostaphin-resistant S. aureus variants had no zones of inhibition. In MBC assays, concentrations of lysostaphin ranging from 0.16 microg/ml to 2.5 microg/ml were found to cause a 3 log or greater drop from the initial CFU of S. aureus within 30 min for all strains tested.

Lysostaphin disrupts Staphylococcus aureus and Staphylococcus epidermidis biofilms on artificial surfaces.

Staphylococci often form biofilms, sessile communities of microcolonies encased in an extracellular matrix that adhere to biomedical implants or damaged tissue. Infections associated with biofilms are difficult to treat, and it is estimated that sessile bacteria in biofilms are 1,000 to 1,500 times more resistant to antibiotics than their planktonic counterparts. This antibiotic resistance of biofilms often leads to the failure of conventional antibiotic therapy and necessitates the removal of infected devices. Lysostaphin is a glycylglycine endopeptidase which specifically cleaves the pentaglycine cross bridges found in the staphylococcal peptidoglycan. Lysostaphin kills Staphylococcus aureus within minutes (MIC at which 90% of the strains are inhibited [MIC(90)], 0.001 to 0.064 microg/ml) and is also effective against Staphylococcus epidermidis at higher concentrations (MIC(90), 12.5 to 64 microg/ml). The activity of lysostaphin against staphylococci present in biofilms compared to those of other antibiotics was, however, never explored. Surprisingly, lysostaphin not only killed S. aureus in biofilms but also disrupted the extracellular matrix of S. aureus biofilms in vitro on plastic and glass surfaces at concentrations as low as 1 microg/ml. Scanning electron microscopy confirmed that lysostaphin eradicated both the sessile cells and the extracellular matrix of the biofilm. This disruption of S. aureus biofilms was specific for lysostaphin-sensitive S. aureus, as biofilms of lysostaphin-resistant S. aureus were not affected. High concentrations of oxacillin (400 microg/ml), vancomycin (800 microg/ml), and clindamycin (800 microg/ml) had no effect on the established S. aureus biofilms in this system, even after 24 h. Higher concentrations of lysostaphin also disrupted S. epidermidis biofilms.