Comparative Secretome Analyses of Three Bacillus anthracis Strains with Variant Plasmid Contents

Abstract

Bacillus anthracis, the causative agent of anthrax, secretes numerous proteins into the extracellular environment during infection. A comparative proteomic approach was employed to elucidate the differences among the extracellular proteomes (secretomes) of three isogenic strains of B. anthracis that differed solely in their plasmid contents. The strains utilized were the wild-type virulent B. anthracis RA3 (pXO1 ؉ pXO2 ؉ ) and its two nonpathogenic derivative strains: the toxigenic, nonencapsulated RA3R (pXO1 ؉ pXO2 ؊ ) and the totally cured, nontoxigenic, nonencapsulated RA3:00 (pXO1 ؊ pXO2 ؊ ). Comparative proteomics using two-dimensional gel electrophoresis followed by computer-assisted gel image analysis was performed to reveal unique, up-regulated, or down-regulated secretome proteins among the strains. In total, 57 protein spots, representing 26 different proteins encoded on the chromosome or pXO1, were identified by peptide mass fingerprinting. S-layer-derived proteins, such as Sap and EA1, were most frequently observed. Many sporulation-associated enzymes were found to be overexpressed in strains containing pXO1؉ . This study also provides evidence that pXO2 is necessary for the maximal expression of the pXO1-encoded toxins lethal factor (LF), edema factor (EF), and protective antigen (PA). Several newly identified putative virulence factors were observed; these include enolase, a high-affinity zinc uptake transporter, the peroxide stress-related alkyl hydroperoxide reductase, isocitrate lyase, and the cell surface protein A.Bacillus anthracis, the etiologic agent of anthrax, is a grampositive, rod-shaped, nonmotile facultative anaerobic and sporeforming bacterium (35). Anthrax is a disease of wildlife, livestock, and humans that begins when spores of B. anthracis enter a host, are engulfed by macrophages, germinate, and then cause septicemia. This pathogen's virulence, along with the spore's resistance to adverse environmental conditions and facility to be weaponized, has made it a significant agent of bioterrorism (1).In addition to a 5.2-Mb circular chromosome, fully virulent strains of B. anthracis harbor two plasmids: pXO1 and pXO2 (35). The pXO1 plasmid (181.6 kb) encodes the toxins lethal factor (LF) and edema factor (EF) and the protective antigen (PA) responsible for the translocation of EF and LF into the host's cytosol (35). The cascade of events leading to toxin entry into host cells has been described (7). The genes encoding LF, EF, and PA are designated lef, cya, and pagA, respectively (8,50,63). Proteins required for a proteinous capsule biosynthesis (CapBCA) and depolymerization (Dep) are encoded on pXO2 (94.8 kb) (29,60). Strains lacking either or both plasmids are attenuated in most animal hosts (22). The transcriptions of lef, cya, pagA, and capB have all been shown to be coordinately induced by the presence of bicarbonate-CO 2 , while temperature has been shown to be important for toxin, but not for CapB, production (55). High CO 2 tension is believed to simulate conditions encountere...

Differential Proteomic Analysis of theBacillus anthracisSecretome: Distinct Plasmid and Chromosome CO2-Dependent Cross Talk Mechanisms Modulate Extracellular Proteolytic Activities

“…Several PA subfragments, representing Ͼ10% of total protein visualized on the 2-DE gel, could be detected (Fig. 3, anthracis culture supernatants has been observed in previous studies (78,146). The other toxin components, LF and EF, were detected in much smaller amounts, in agreement with previous studies (81).…”

“…Both pXO1-90 and pXO1-130 were documented in a previous report from our laboratory to be immunogens expressed in vivo, and they represent potential anthrax vaccine candidates (7). These two proteins were also identified in the supernatant of B. anthracis cultures under high CO 2 tension by Lamonica and coworkers (78). These proteins may be involved in bacterial pathogenesis: detection of the protein pXO1-90 (an SLH-exhibiting protein of unknown function) is in good agreement with the observation that it is transcriptionally activated by the virulence regulator AtxA (17).…”

“…Here, we document an extended proteomic study, focusing on B. anthracis secreted proteins, which expands the data set of expressed B. anthracis proteins from both virulent and nonvirulent strains (6,25,41,50,64,78,85,144). Based on identification of more than 400 two-dimensional electrophoresis (2-DE)-separated protein spots, we report the expression of 64 proteins which represent the most abundant B. anthracis secreted proteins, many of which resemble factors involved in the virulence of other pathogens.…”

“…The availability of the B. anthracis genomic DNA sequence (109,121) paved the way for high-throughput genomic, transcriptomic, and proteomic analyses of B. anthracis (6,7,8,17,25,41,50,64,78,85,121,144) in an effort to elucidate pathogenicity mechanisms by identification of novel virulence factors or in search for specific therapeutic and/or diagnostic targets. Indeed, bioinformatic surveys of the B. anthracis genome (7,8,121) suggested that proteins other than PA, LF, and EF, may participate in anthrax pathogenesis.…”

Is it possible to consider «kinetomics» and «argyromics» as novel trends of functional morphology and systems biology of parasitic and nonparasitic protozoa? a brief analytical review

“…As an example of practical importance we consider a comparative projection of descriptors of the secreting cells (or tissues) to the subspace of the micro-colorimetric measurement results of the Mallory stained sample analysis in the frame of the secretome expression concept (Zwickl, 2005;Martinez, 2006) and comparative "secretomics" (Lamonica, 2005;Sibbald, 2006) which can also be used for the parasitic protozoa-related phenomena (Przyborski, 2004). A less trivial example is the relation between metallomics or microelementomics (Li, 2008) and an old Mallory method of lead and copper determination (Mallory, 1939), because to the best of our knowledge the methods of cellular metallomic and elementomic mapping have not yet been developed.…”