Redox proteomic study of Bacillus cereus thiol proteome during fermentative anaerobic growth.

BACKGROUND: Bacillus cereus is a notorious foodborne pathogen, which can grow under anoxic conditions. Anoxic growth is supported by endogenous redox metabolism, for which the thiol redox proteome serves as an interface. Here, we studied the cysteine (Cys) proteome dynamics of B. cereus ATCC 14579 cells grown under fermentative anoxic conditions. We used a quantitative thiol trapping method combined with proteomics profiling. RESULTS: In total, we identified 153 reactive Cys residues in 117 proteins participating in various cellular processes and metabolic pathways, including translation, carbohydrate metabolism, and stress response. Of these reactive Cys, 72 were detected as reduced Cys. The B. cereus Cys proteome evolved during growth both in terms of the number of reduced Cys and the Cys-containing proteins identified, reflecting its growth-phase-dependence. Interestingly, the reduced status of the B. cereus thiol proteome increased during growth, concomitantly to the decrease of extracellular oxidoreduction potential. CONCLUSIONS: Taken together, our data show that the B. cereus Cys proteome during unstressed fermentative anaerobic growth is a dynamic entity and provide an important foundation for future redox proteomic studies in B. cereus and other organisms.

Cysteine Proteome Reveals Response to Endogenous Oxidative Stress in Bacillus cereus.

At the end of exponential growth, aerobic bacteria have to cope with the accumulation of endogenous reactive oxygen species (ROS). One of the main targets of these ROS is cysteine residues in proteins. This study uses liquid chromatography coupled to high-resolution tandem mass spectrometry to detect significant changes in protein abundance and thiol status for cysteine-containing proteins from Bacillus cereus during aerobic exponential growth. The proteomic profiles of cultures at early-, middle-, and late-exponential growth phases reveals that (i) enrichment in proteins dedicated to fighting ROS as growth progressed, (ii) a decrease in both overall proteome cysteine content and thiol proteome redox status, and (iii) changes to the reduced thiol status of some key proteins, such as the transition state transcriptional regulator AbrB. Taken together, our data indicate that growth under oxic conditions requires increased allocation of protein resources to attenuate the negative effects of ROS. Our data also provide a strong basis to understand the response mechanisms used by B. cereus to deal with endogenous oxidative stress.

B cells response directed against Cut4 and CFP21 lipolytic enzymes in active and latent tuberculosis infections.

BACKGROUND: Better understanding of the immune response directed against Mycobacterium tuberculosis (Mtb) is critical for development of vaccine strategies and diagnosis tests. Previous studies suggested that Mtb enzymes involved in lipid metabolism, are associated with persistence and/or reactivation of dormant bacilli. METHODS: Circulating antibodies secreting cells (ASCs), memory B cells, and antibodies directed against Cut4 (Rv3452) and CFP21 (Rv1984c) antigens were explored in subjects with either active- or latent-tuberculosis (LTB), and in Mtb-uninfected individuals. RESULTS: Circulating anti-Cut4 ASCs were detected in 11/14 (78.6%) subjects from the active TB group vs. 4/17 (23.5%) from the LTB group (p = 0.001). Anti-CFP21 ASCs were found in 11/14 (78.6%) active TB vs. in 5/17 (29.4%) LTB cases (p = 0.01). Circulating anti-Cut4 and anti-CFP21 ASCs were not detected in 38 Mtb uninfected controls. Memory B cells directed against either Cut4 or CFP21 were identified in 8/11 (72.7%) and in 9/11 (81.8%) subjects with LTB infection, respectively, and in 2/6 Mtb uninfected individuals (33.3%). High level of anti-Cut4 and anti-CFP21 IgG were observed in active TB cases. CONCLUSION: Circulating IgG SCs directed against Cut4 or CFP21 were mostly detected in patients presenting an active form of the disease, suggesting that TB reactivation triggers an immune response against these two antigens.

Identification of residues involved in substrate specificity and cytotoxicity of two closely related cutinases from Mycobacterium tuberculosis.

The enzymes belonging to the cutinase family are serine enzymes active on a large panel of substrates such as cutin, triacylglycerols, and phospholipids. In the M. tuberculosis H37Rv genome, seven genes coding for cutinase-like proteins have been identified with strong immunogenic properties suggesting a potential role as vaccine candidates. Two of these enzymes which are secreted and highly homologous, possess distinct substrates specificities. Cfp21 is a lipase and Cut4 is a phospholipase A2, which has cytotoxic effects on macrophages. Structural overlay of their three-dimensional models allowed us to identify three areas involved in the substrate binding process and to shed light on this substrate specificity. By site-directed mutagenesis, residues present in these Cfp21 areas were replaced by residues occurring in Cut4 at the same location. Three mutants acquired phospholipase A1 and A2 activities and the lipase activities of two mutants were 3 and 15 fold greater than the Cfp21 wild type enzyme. In addition, contrary to mutants with enhanced lipase activity, mutants that acquired phospholipase B activities induced macrophage lysis as efficiently as Cut4 which emphasizes the relationship between apparent phospholipase A2 activity and cytotoxicity. Modification of areas involved in substrate specificity, generate recombinant enzymes with higher activity, which may be more immunogenic than the wild type enzymes and could therefore constitute promising candidates for antituberculous vaccine production.

Regulation of cel genes of C. cellulolyticum: identification of GlyR2, a transcriptional regulator regulating cel5D gene expression.

Transcription and expression regulation of some individual cel genes (cel5A, cel5I, cel5D and cel44O) of Clostridium cellulolyticum were investigated. Unlike the cip-cel operon, these genes are transcribed as monocistronic units of transcription, except cel5D. The location of the transcription initiation sites was determined using RT-PCR and the mRNA 5'-end extremities were detected using primer extension experiments. Similarly to the cip-cel operon, cel5A and cel5I expressions are regulated by a carbon catabolite repression mechanism, whereas cel44O and cel5D expressions do not seem to be submitted to this regulation. The role of the putative transcriptional regulator GlyR2 in the regulation of cel5D expression was investigated. The recombinant protein GlyR2 was produced and was shown to bind in vitro to the cel5D and glyR2 promoter regions, suggesting that besides regulating its own expression, GlyR2 may regulate cel5D expression. To test this hypothesis in vivo, an insertional glyR2 mutant was generated and the effect of this disruption on cel5D expression was evaluated. Levels of cel5D mRNAs in the mutant were 16 fold lower than that of the wild-type strain suggesting that GlyR2 acts as an activator of cel5D expression.

MmPPOX inhibits Mycobacterium tuberculosis lipolytic enzymes belonging to the hormone-sensitive lipase family and alters mycobacterial growth.

Lipid metabolism plays an important role during the lifetime of Mycobacterium tuberculosis, the causative agent of tuberculosis. Although M. tuberculosis possesses numerous lipolytic enzymes, very few have been characterized yet at a biochemical/pharmacological level. This study was devoted to the M. tuberculosis lipolytic enzymes belonging to the Hormone-Sensitive Lipase (HSL) family, which encompasses twelve serine hydrolases closely related to the human HSL. Among them, nine were expressed, purified and biochemically characterized using a broad range of substrates. In vitro enzymatic inhibition studies using the recombinant HSL proteins, combined with mass spectrometry analyses, revealed the potent inhibitory activity of an oxadiazolone compound, named MmPPOX. In addition, we provide evidence that MmPPOX alters mycobacterial growth. Overall, these findings suggest that the M. tuberculosis HSL family displays important metabolic functions, thus opening the way to further investigations linking the involvement of these enzymes in mycobacterial growth.

Mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis.

BACKGROUND: New diagnosis tests are urgently needed to address the global tuberculosis (TB) burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452) as new markers in the serodiagnosis of active TB. METHODS: Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. RESULTS: A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. CONCLUSION: These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent biomarkers providing high sensitivity and specificity levels for the immunodiagnosis of active TB.

The omp50 gene is transcriptionally controlled by a temperature-dependent mechanism conserved among thermophilic Campylobacter species.

The thermophilic Campylobacters are enteropathogenic for humans. We recently showed that Omp50 is a Campylobacter species-specific porin produced in Campylobacter jejuni and Campylobacter lari but not in Campylobacter coli. In the present study, we investigated regulation of the omp50 gene and found that its expression in C. jejuni was temperature-dependent, but independent of growth phase or medium viscosity. The use of RT-PCR and omp50::lacZ fusions showed that growth temperature control occurred at the transcriptional level. The promoter and the coding sequence were cloned in an Escherichia coli-Campylobacter shuttle plasmid and transferred to E. coli and to a C. jejuni Omp50-deficient strain. Regulation of omp50 gene expression by growth temperature was observed in the recombinant C. jejuni strain, but not in E. coli. The same regulation was also observed in wild-type C. lari strains and in a C. coli strain supplemented by the plasmid, suggesting that omp50 expression is controlled by a mechanism conserved among Campylobacter species.

Chromosomal His-tagging: an alternative approach to membrane protein purification.

Membrane proteins are of keen interest to structural biologists, as they are known to act as receptors, adhesins, sensors, transporters, and signal-transducers of living cells. During the past few decades, the efforts made to study the bacterial membrane proteins have been impaired by the problems encountered during the production and purification of native proteins. Herein we demonstrate that the Campylobacter jejuni CadF protein, which was isolated using a novel purification strategy, exhibits biological activity as evidenced by channel activity in lipid bilayers. CadF, an E. coli OmpA-like protein, facilitates the binding of C. jejuni to the extracellular matrix component, fibronectin.

Use of the omp50 gene for identification of Campylobacter species by PCR.

We studied the prevalence of the omp50 gene and the Omp50 protein in Campylobacter strains. Immunodetection assays and DNA-DNA hybridizations showed that most C. coli strains tested were negative and most C. jejuni and C. lari strains tested were positive. A PCR assay was developed, using the omp50 gene as a species-specific target. We propose a combination of a hippurate test and an omp50 assay to perform identification of Campylobacter species.

Environmental regulation of Campylobacter jejuni major outer membrane protein porin expression in Escherichia coli monitored by using green fluorescent protein.

Porins allow exchanges between bacteria and their environment. In the gram-negative food-borne pathogen Campylobacter jejuni two porins, major outer membrane protein (MOMP) and Omp50, have been identified. MOMP is synthesized at a very high level under laboratory culture conditions, suggesting that its promoter functions very efficiently under these conditions. In Campylobacter samples, we observed that MOMP porin expression increased at a high temperature (42 degrees C) or a high pH (pH 8.5) compared to expression at a low temperature (31 degrees C) or an acidic pH (pH 5.5). To study the regulation of MOMP expression at the transcriptional level, we constructed an momp-gfp fusion in which gfp expression was put under the control of the momp promoter. Interestingly, we observed the same pattern of regulation in Escherichia coli, as monitored by green fluorescent protein production, that was found in CAMPYLOBACTER: The ranges of pH and temperature tested are physiologically relevant, because they can be found in the digestive tracts of both birds and humans, which are both colonized by CAMPYLOBACTER: Our results suggest that a component of the regulatory mechanism is conserved in C. jejuni and E. coli. However, medium osmolarity and sodium salicylate did not have a significant effect on C. jejuni momp promoter activity in E. coli, suggesting that major regulatory elements of E. coli porin expression do not participate in MOMP regulation. In contrast, mechanisms involving DNA supercoiling may be involved, as shown by DNA gyrase inhibition assays. These findings are a step towards determining the role of outer membrane proteins in the adaptation of C. jejuni to its environment.