Antigen Discovery for Next-Generation Pertussis Vaccines Using Immunoproteomics and Transposon-Directed Insertion Sequencing.

BACKGROUND: Despite high vaccination rates, the United States has experienced a resurgence in reported cases of pertussis after switching to the acellular pertussis vaccine, indicating a need for improved vaccines that enhance infection control. METHODS: Bordetella pertussis antigens recognized by convalescent-baboon serum and nasopharyngeal wash were identified by immunoproteomics and their subcellular localization predicted. Genes essential or important for persistence in the baboon airway were identified by transposon-directed insertion-site sequencing (TraDIS) analysis. RESULTS: In total, 314 B. pertussis antigens were identified by convalescent baboon serum and 748 by nasopharyngeal wash. Thirteen antigens were identified as immunogenic in baboons, essential for persistence in the airway by TraDIS, and membrane-localized: BP0840 (OmpP), Pal, OmpA2, BP1485, BamA, Pcp, MlaA, YfgL, BP2197, BP1569, MlaD, ComL, and BP0183. CONCLUSIONS: The B. pertussis antigens identified as immunogenic, essential for persistence in the airway, and membrane-localized warrant further investigation for inclusion in vaccines designed to reduce or prevent carriage of bacteria in the airway of vaccinated individuals.

The extended N-terminus of Mycobacterium smegmatis RecX potentiates its ability to antagonize RecA functions.

The members of the RecX family of proteins have a unique capacity to regulate the catalytic activities of RecA/Rad51 proteins in both prokaryotic and eukaryotic organisms. However, our understanding of the functional roles of RecX in pathogenic and non-pathogenic mycobacteria has been limited by insufficient knowledge of the molecular mechanisms of its activity and regulation. Moreover, the significance of a unique 14 amino acid N-terminal extension in Mycobacterium smegmatis RecX (MsRecX) to its function remains unknown. Here, we advance our understanding of the antagonistic roles of mycobacterial RecX proteins and the functional significance of the extended N-terminus of MsRecX. The full-length MsRecX acts as an antagonist of RecA, negatively regulating RecA promoted functions, including DNA strand exchange, LexA cleavage and ATP hydrolysis, but not binding of ATP. The N-terminally truncated MsRecX variants retain the RecA inhibitory activity, albeit with lower efficiencies compared to the full-length protein. Perhaps most importantly, direct visualization of RecA nucleoprotein filaments, which had been incubated with RecX proteins, showed that they promote disassembly of nucleoprotein filaments primarily within the filaments. In addition, interaction of RecX proteins with the RecA nucleoprotein filaments results in the formation of stiff and irregularly shaped nucleoprotein filaments. Thus, these findings add an additional mechanism by which RecX disassembles RecA nucleoprotein filaments. Overall, this study provides strong evidence for the notion that the N-terminal 14 amino acid region of MsRecX plays an important role in the negative regulation of RecA functions and new insights into the molecular mechanism underlying RecX function.

Microbiota of MR1 deficient mice confer resistance against Clostridium difficile infection.

Clostridium difficile (Cd) infection (CDI) typically occurs after antibiotic usage perturbs the gut microbiota. Mucosa-associated invariant T cells (MAIT) are found in the gut and their development is dependent on Major histocompatibility complex-related protein 1 (MR1) and the host microbiome. Here we were interested in determining whether the absence of MR1 impacts resistance to CDI. To this end, wild-type (WT) and MR1-/- mice were treated with antibiotics and then infected with Cd spores. Surprisingly, MR1-/- mice exhibited resistance to Cd colonization. 16S rRNA gene sequencing of feces revealed inherent differences in microbial composition. This colonization resistance was transferred from MR1-/- to WT mice via fecal microbiota transplantation, suggesting that MR1-dependent factors influence the microbiota, leading to CDI susceptibility.

Elucidating the functional role of Mycobacterium smegmatis recX in stress response.

The RecX protein has attracted considerable interest because the recX mutants exhibit multiple phenotypes associated with RecA functions. To further our understanding of the functional relationship between recA and recX, the effect of different stress treatments on their expression profiles, cell yield and viability were investigated. A significant correlation was found between the expression of Mycobacterium smegmatis recA and recX genes at different stages of growth, and in response to different stress treatments albeit recX exhibiting lower transcript and protein abundance at the mid-log and stationary phases of the bacterial growth cycle. To ascertain their roles in vivo, a targeted deletion of the recX and recArecX was performed in M. smegmatis. The growth kinetics of these mutant strains and their sensitivity patterns to different stress treatments were assessed relative to the wild-type strain. The deletion of recA affected normal cell growth and survival, while recX deletion showed no significant effect. Interestingly, deletion of both recX and recA genes results in a phenotype that is intermediate between the phenotypes of the ΔrecA mutant and the wild-type strain. Collectively, these results reveal a previously unrecognized role for M. smegmatis recX and support the notion that it may regulate a subset of the yet unknown genes involved in normal cell growth and DNA-damage repair.

The Anionic Phospholipids in the Plasma Membrane Play an Important Role in Regulating the Biochemical Properties and Biological Functions of RecA Proteins.

Escherichia coli RecA (EcRecA) forms discrete foci that cluster at cell poles during normal growth, which are redistributed along the filamented cell axis upon induction of the SOS response. The plasma membrane is thought to act as a scaffold for EcRecA foci, thereby playing an important role in RecA-dependent homologous recombination. In addition, in vivo and in vitro studies demonstrate that EcRecA binds strongly to the anionic phospholipids. However, there have been almost no data on the association of mycobacterial RecA proteins with the plasma membrane and the effects of membrane components on their function. Here, we show that mycobacterial RecA proteins specifically interact with phosphatidylinositol and cardiolipin among other anionic phospholipids; however, they had no effect on the ability of RecA proteins to bind single-stranded DNA. Interestingly, phosphatidylinositol and cardiolipin impede the DNA-dependent ATPase activity of RecA proteins, although ATP binding is not affected. Furthermore, the ability of RecA proteins to promote DNA strand exchange is not affected by anionic phospholipids. Strikingly, anionic phospholipids suppress the RecA-stimulated autocatalytic cleavage of the LexA repressor. The Mycobacterium smegmatis RecA foci localize to the cell poles during normal growth, and these structures disassemble and reassemble into several foci along the cell after the induction of DNA damage. Taken together, these data support the notion that the interaction of RecA with cardiolipin and phosphatidylinositol, the major anionic phospholipids of the mycobacterial plasma membrane, may be physiologically relevant, as they provide a scaffold for RecA storage and may regulate recombinational DNA repair and the SOS response.

The second messenger cyclic di-AMP negatively regulates the expression of Mycobacterium smegmatis recA and attenuates DNA strand exchange through binding to the C-terminal motif of mycobacterial RecA proteins.

Cyclic di-GMP and cyclic di-AMP are second messengers produced by a wide variety of bacteria. They influence bacterial cell survival, biofilm formation, virulence and bacteria-host interactions. However, many of their cellular targets and biological effects are yet to be determined. A chemical proteomics approach revealed that Mycobacterium smegmatis RecA (MsRecA) possesses a high-affinity cyclic di-AMP binding activity. We further demonstrate that both cyclic di-AMP and cyclic di-GMP bind specifically to the C-terminal motif of MsRecA and Mycobacterium tuberculosis RecA (MtRecA). Escherichia coli RecA (EcRecA) was devoid of cyclic di-AMP binding but have cyclic di-GMP binding activity. Notably, cyclic di-AMP attenuates the DNA strand exchange promoted by MsRecA as well as MtRecA through the disassembly of RecA nucleoprotein filaments. However, the structure and DNA strand exchange activity of EcRecA nucleoprotein filaments remain largely unaffected. Furthermore, M. smegmatis ΔdisA cells were found to have undetectable RecA levels due to the translational repression of recA mRNA. Consequently, the ΔdisA mutant exhibited enhanced sensitivity to DNA-damaging agents. Altogether, this study points out the importance of sequence diversity among recA genes, the role(s) of cyclic di-AMP and reveals a new mode of negative regulation of recA gene expression, DNA repair and homologous recombination in mycobacteria.