Application of In Vivo Induced Antigen Technology (IVIAT) to Bacillus anthracis

In vivo induced antigen technology (IVIAT) is an immuno-screening technique that identifies bacterial antigens expressed during infection and not during standard in vitro culturing conditions. We applied IVIAT to Bacillus anthracis and identified PagA, seven members of a N-acetylmuramoyl-L-alanine amidase autolysin family, three P60 family lipoproteins, two transporters, spore cortex lytic protein SleB, a penicillin binding protein, a putative prophage holin, respiratory nitrate reductase NarG, and three proteins of unknown function. Using quantitative real-time PCR comparing RNA isolated from in vitro cultured B. anthracis to RNA isolated from BALB/c mice infected with virulent Ames strain B. anthracis, we confirmed induced expression in vivo for a subset of B. anthracis genes identified by IVIAT, including L-alanine amidases BA3767, BA4073, and amiA (pXO2-42); the bacteriophage holin gene BA4074; and pagA (pXO1-110). The exogenous addition of two purified putative autolysins identified by IVIAT, N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446, to vegetative B. anthracis cell suspensions induced a species-specific change in bacterial morphology and reduction in viable bacterial cells. Many of the proteins identified in our screen are predicted to affect peptidoglycan re-modeling, and our results support significant cell wall structural remodeling activity during B. anthracis infection. Identification of L-alanine amidases with B. anthracis specificity may suggest new potential therapeutic targets

Virulence of Staphylococcus aureus Small Colony Variants in the Caenorhabditis elegans Infection Model

Small colony variants (SCVs) of Staphylococcus aureus are slow-growing morphological variants that have been implicated in persistent, relapsing, and antibiotic-resistant infections. The altered phenotype of SCVs in most strains has been attributed to defects in electron transport due to mutations in hemin or menadione biosynthesis. The pathogenic capacity of SCVs compared to phenotypically normal strains is variable depending on the attribute examined, with some studies showing reduced virulence of SCVs and others demonstrating normal or heightened virulence. Recently, the nematode Caenorhabditis elegans has been successfully employed as an alternative host to investigate virulence mechanisms of a variety of bacterial pathogens, including S. aureus. In this study, we show that clinical SCVs as well as hemB- and menD-deficient mutants of S. aureus are greatly reduced in virulence in the C. elegans infection model

Identification of In Vivo-Induced Bacterial Protein Antigens during Human Infection with Salmonella enterica Serovar Typhi

We applied an immunoscreening technique, in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed during human infection with Salmonella enterica serovar Typhi, the cause of typhoid fever. We were able to assign a functional classification to 25 of 35 proteins identified by IVIAT. Of these 25, the majority represent proteins with known or potential roles in the pathogenesis of S. enterica. These include proteins implicated in fimbrial structure and biogenesis, antimicrobial resistance, heavy metal transport, bacterial adhesion, and extracytoplasmic substrate trafficking as well as secreted hydrolases. The 10 remaining antigens represent proteins with unknown functions. Of the 35 identified antigens, four had no immunoreactivity when probed with control sera from individuals never exposed to serovar Typhi organisms; these four included PagC, TcfB, and two antigens of unknown function encoded by STY0860 and STY3683. PagC is a virulence factor known to be upregulated in vivo in S. enterica serovar Typhimurium infection of mice. TcfB is the major structural subunit of a fimbrial operon found in serovar Typhi with no homolog in serovar Typhimurium organisms. By examining differential immunoreactivities in acute- versus convalescent-phase human serum samples, we found specific anti-PagC and anti-TcfB immunoglobulin G responses in patients with serovar Typhi bacteremia. Serovar Typhi antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and they may have diagnostic, therapeutic, or preventive uses

<i>B. anthracis</i> gene products identified by IVIAT.

<p><i>B. anthracis</i> gene products identified by IVIAT.</p

mRNA expression profiles during in vitro and in vivo growth of IVIAT-identified and paralog/similar <i>B. anthracis</i> genes.

<p>Quantitative RT-PCR was performed on RNA recovered from in vitro grown <i>B. anthracis</i> (Ames strain cells grown in BHI and air; late-log phase) and compared to RNA recovered from mice infected with vegetative Ames strain <i>B. anthracis</i> via intravenous injection. RNA from mice was isolated from spleens 18 hr post-infection. Transcript numbers on the Y-axis are normalized against 16S rRNA. Displayed are gene profiles of <i>B. anthracis</i> genes up-regulated in vivo compared to in vitro. In bar graphs, expression levels represent mean values; error bars represent standard deviations (SD). In summary table, expression given as mRNA transcript copies (× 10⁻⁵) per copy of 16S rRNA.</p

<i>B. anthracis</i> species-specific autolysis effects following addition of exogenous putative autolysins N-acetylmuramoyl-L-alanine amidases BA0485 and BA2446: (A) reduction in A₅₉₅ optical density, (B) reduction in colony forming units, and (C, D) morphological changes of <i>B. cereus</i> (C) or <i>B. anthracis</i> (D).

<p><i>B. anthracis</i> (Sterne strain), <i>B. cereus</i> (ATCC 14579), <i>B. subtilis</i> (168), and <i>E. coli</i> (DH5α) vegetative cells were resuspended in 20 mM sodium phosphate buffer containing BA0485 and BA2446 (final concentration 2 uM) or buffer alone (BA0485, pH 7.0; BA2446, pH 8.7). (A) A₅₉₅ readings were recorded every 2 min. and reported as a percentage of starting A₅₉₅. Buffer-only controls were not different for cultures containing <i>B. cereus, B. subtilis,</i> or <i>E. coli</i>. (B) Dilutions of cells were plated prior to addition of BA0485 and BA2446 proteins and then again following 20 min of incubation with buffer alone or buffer containing exogenous BA0485 or BA2446, and CFU are reported as percentage of the starting CFU. (C, D) BA0485, BA2446, or buffer alone were added to cell suspensions of <i>B. anthracis</i>, <i>B. cereus, B. subtilis,</i> and <i>E. coli</i>, and incubated for 20 min. Bacterial morphological changes were only evident following protein addition to <i>B. anthracis</i> (D); representative <i>B. cereus</i> samples are shown (C). Gram stains were performed and all images were captured at 1000×.</p

<i>Bacillus</i> strains used in this study.

<p><i>Bacillus</i> strains used in this study.</p