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Detection of microbial antigens in clinical samples can lead to rapid diagnosis of an infection and administration of appropriate therapeutics. A major barrier in diagnostics development is determining which of the potentially hundreds or thousands of antigens produced by a microbe are actually present in patient samples in detectable amounts against a background of innumerable host proteins. In this report, we describe a strategy, termed in vivo microbial antigen discovery (InMAD), that we used to identify circulating bacterial antigens. This technique starts with "InMAD serum," which is filtered serum that has been harvested from BALB/c mice infected with a bacterial pathogen. The InMAD serum, which is free of whole bacterial cells, is used to immunize syngeneic BALB/c mice. The resulting " InMAD immune serum" contains antibodies specific for the soluble microbial antigens present in sera from the infected mice. The InMAD immune serum is then used to probe blots of bacterial lysates or bacterial proteome arrays. Bacterial antigens that are reactive with the InMAD immune serum are precisely the antigens to target in an antigen immunoassay. By employing InMAD, we identified multiple circulating antigens that are secreted or shed during infection using Burkholderia pseudomallei and Francisella tularensis as model organisms. Potential diagnostic targets identified by the InMAD approach included bacterial proteins, capsular polysaccharide, and lipopolysaccharide. The InMAD technique makes no assumptions other than immunogenicity and has the potential to be a broad discovery platform to identify diagnostic targets from microbial pathogens. IMPORTANCE Effective treatment of microbial infection is critically dependent on early diagnosis and identification of the etiological agent. One means for rapid diagnosis is immunoassay for antigens that are shed into body fluids during infection. Immunoassays can be inexpensive, rapid, and adaptable to a point-of-care format. A major impediment to immunoassay for diagnosis of infectious disease is identification of appropriate antigen targets. This report describes a strategy that can be used for identification of microbial antigens that are shed into serum during infection by the biothreats Burkholderia pseudomallei and Francisella tularensis. Termed InMAD (in vivo microbial antigen discovery), the strategy has the potential for application to a broad spectrum of microbial pathogens.

In the current study, we examined the regulatory interactions of a serine/threonine phosphatase (BA-Stp1), serine/threonine kinase (BA-Stk1) pair in Bacillus anthracis. B. anthracis STPK101, a null mutant lacking BA-Stp1 and BA-Stk1, was impaired in its ability to survive within macrophages, and this correlated with an observed reduction in virulence in a mouse model of pulmonary anthrax. Biochemical analyses confirmed that BA-Stp1 is a PP2C phosphatase and dephosphorylates phosphoserine and phosphothreonine residues. Treatment of BA-Stk1 with BA-Stp1 altered BA-Stk1 kinase activity, indicating that the enzymatic function of BA-Stk1 can be influenced by BA-Stp1 dephosphorylation. Using a combination of mass spectrometry and mutagenesis approaches, three phosphorylated residues, T165, S173, and $214, in BA-Stk1 were identified as putative regulatory targets of BA-Stp1. Further analysis found that T165 and S173 were necessary for optimal substrate phosphorylation, while $214 was necessary for complete ATP hydrolysis, autophosphorylation, and substrate phosphorylation. These findings provide insight into a previously undescribed Stp/Stk pair in B. anthracis. doi: 10.1128/JB.01221-09

Mycobacterium abscessus is an increasingly important cause of human disease; however, virulence determinants are largely uncharacterized. Previously, it was demonstrated that a rough, wild-type human clinical isolate (390R) causes persistent, invasive infection, while a smooth isogenic mutant (390S) has lost this capability. During serial passage of 390S, a spontaneous rough revertant was obtained, which was named 390V. This revertant regained the ability to cause persistent, invasive infection in human monocytes and the lungs of mice. Glycopeptidolipid (GPL), which plays a role in environmental colonization, was present in abundance in the cell wall of 390S, and was associated with sliding motility and biofilm formation. In contrast, a marked reduction in the amount of GPL in the cell wall of 390R and 390V was correlated with cord formation, a property associated with mycobacterial virulence. These results indicate that the ability to switch between smooth and rough morphologies may allow M. abscessus to transition between a colonizing phenotype and a more virulent, invasive form.

Abstract Background The resolution of inflammatory responses in the lung has not been described in detail and the role of specific cytokines influencing the resolution process is largely unknown. Methods The present study was designed to describe the resolution of inflammation from 3 h through 90 d following an acute injury by a single intratracheal instillation of F344/N rats with LPS. We documented the inflammatory cell types and cytokines found in the bronchoalveolar lavage fluid (BALF), and epithelial changes in the axial airway and investigated whether IL-18 may play a role in the resolution process by reducing its levels with anti-IL-18 antibodies. Results Three major stages of inflammation and resolution were observed in the BALF during the resolution. The first stage was characterized by PMNs that increased over 3 h to 1 d and decreased to background levels by d 6–8. The second stage of inflammation was characterized by macrophage influx reaching maximum numbers at d 6 and decreasing to background levels by d 40. A third stage of inflammation was observed for lymphocytes which were elevated over d 3–6. Interestingly, IL-18 and IL-9 levels in the BALF showed a cyclic pattern with peak levels at d 4, 8, and 16 while decreasing to background levels at d 1–2, 6, and 12. Depletion of IL-18 caused decreased PMN numbers at d 2, but no changes in inflammatory cell number or type at later time points. Conclusion These data suggest that IL-18 plays a role in enhancing the LPS-induced neutrophilic inflammation of the lung, but does not affect the resolution of inflammation.

Yersinia pestis, the causative agent of plague, can be transmitted by infected flea bite or inhaled aerosol. Both routes of infection have a high mortality rate, and pneumonic infections of Y. pestis represent a significant concern as a tool of bioterrorism. Understanding the transcriptional program of this pathogen during pulmonary infection should be valuable in understanding plague pathogenesis, as well as potentially offering insights into new vaccines and therapeutics. Toward this goal we developed a long oligonucleotide microarray to the plague bacillus and evaluated the expression profiles of Y. pestis in vitro and in the mouse pulmonary infection model in vivo. The in vitro analysis compared expression patterns at 27 versus 37°C, as a surrogate of the transition from the flea to the mammalian host. The in vivo analysis used intranasal challenge to the mouse lung. By amplifying the Y. pestis RNA from individual mouse lungs we were able to map the transcriptional profile of plague at postinfection days 1 to 3. Our data present a very different transcriptional profile between in vivo and in vitro expression, suggesting Y. pestis responds to a variety of host signals during infection. Of note was the number of genes found in genomic regions with altered %GC content that are upregulated within the mouse lung environment. These data suggest these regions may provide particularly promising targets for both vaccines and therapeutics. [ABSTRACT FROM AUTHOR]

Bacillus anthracis, the agent of anthrax, produces a poly-D-glutamic acid capsule that has been implicated in virulence. Many strains missing pXO2 (96 kb), which harbors the capsule biosynthetic operon capBCAD, but carrying pXO1 (182 kb) that harbors the anthrax toxin genes, are attenuated in animal models. Also, noncapsulated strains are readily phagocytosed by macrophage cell lines, whereas capsulated strains are resistant to phagocytosis. We show that a strain carrying both virulence plasmids but deleted specifically for capBCAD is highly attenuated in a mouse model for inhalation anthrax. The parent strain and capsule mutant initiated germination in the lungs, but the capsule mutant did not disseminate to the spleen. A mutant harboring capBCAD but deleted for the cap regulators acpA and acpB was also significantly attenuated, in agreement with the capsule-negative phenotype during in vitro growth. Surprisingly, an acpB mutant, but not an acpA mutant, displayed an elevated LD50 and reduced ability to disseminate, indicating that acpA and acpB are not true functional homologs and that acpB may play a larger role in virulence than originally suspected. [ABSTRACT FROM AUTHOR]