Similarity to peroxisomal-membrane protein family reveals that Sinorhizobium and Brucella BacA affect lipid-A fatty acids

Sinorhizobium meliloti, a legume symbiont, and Brucella abortus, a phylogenetically related mammalian pathogen, both require the bacterial-encoded BacA protein to establish chronic intracellular infections in their respective hosts. We found that the bacterial BacA proteins share sequence similarity with a family of eukaryotic peroxisomal-membrane proteins, including the human adrenoleukodystrophy protein, required for the efficient transport of verylong- chain fatty acids out of the cytoplasm. This insight, along with the increased sensitivity of BacA-deficient mutants to detergents and cell envelope-disrupting agents, led us to discover that BacA affects the very-long-chain fatty acid (27-OHC28:0 and 29-OHC30:0) content of both Sinorhizobium and Brucella lipid A. We discuss models for how BacA function affects the lipid-A fatty-acid content and why this activity could be important for the establishment of chronic intracellular infections. Originally published Proceedings of the National Academy of Sciences, Vol. 101, No. 14, Apr 200

Brucella abortus Infection of Placental Trophoblasts Triggers Endoplasmic Reticulum Stress-Mediated Cell Death and Fetal Loss via Type IV Secretion System-Dependent Activation of CHOP.

Subversion of endoplasmic reticulum (ER) function is a feature shared by multiple intracellular bacteria and viruses, and in many cases this disruption of cellular function activates pathways of the unfolded protein response (UPR). In the case of infection with Brucella abortus, the etiologic agent of brucellosis, the unfolded protein response in the infected placenta contributes to placentitis and abortion, leading to pathogen transmission. Here we show that B. abortus infection of pregnant mice led to death of infected placental trophoblasts in a manner that depended on the VirB type IV secretion system (T4SS) and its effector VceC. The trophoblast death program required the ER stress-induced transcription factor CHOP. While NOD1/NOD2 expression in macrophages contributed to ER stress-induced inflammation, these receptors did not play a role in trophoblast death. Both placentitis and abortion were independent of apoptosis-associated Speck-like protein containing a caspase activation and recruitment domain (ASC). These studies show that B. abortus uses its T4SS to induce cell-type-specific responses to ER stress in trophoblasts that trigger placental inflammation and abortion. Our results suggest further that in B. abortus the T4SS and its effectors are under selection as bacterial transmission factors.IMPORTANCE Brucella abortus infects the placenta of pregnant cows, where it replicates to high levels and triggers abortion of the calf. The aborted material is highly infectious and transmits infection to both cows and humans, but very little is known about how B. abortus causes abortion. By studying this infection in pregnant mice, we discovered that B. abortus kills trophoblasts, which are important cells for maintaining pregnancy. This killing required an injected bacterial protein (VceC) that triggered an endoplasmic reticulum (ER) stress response in the trophoblast. By inhibiting ER stress or infecting mice that lack CHOP, a protein induced by ER stress, we could prevent death of trophoblasts, reduce inflammation, and increase the viability of the pups. Our results suggest that B. abortus injects VceC into placental trophoblasts to promote its transmission by abortion

Tobacco Smoke Augments Porphyromonas gingivalis - Streptococcus gordonii Biofilm Formation

Smoking is responsible for the majority of periodontitis cases in the US and smokers are more susceptible than non-smokers to infection by the periodontal pathogen Porphyromonas gingivalis. P. gingivalis colonization of the oral cavity is dependent upon its interaction with other plaque bacteria, including Streptococcus gordonii. Microarray analysis suggested that exposure of P. gingivalis to cigarette smoke extract (CSE) increased the expression of the major fimbrial antigen (FimA), but not the minor fimbrial antigen (Mfa1). Therefore, we hypothesized that CSE promotes P. gingivalis-S. gordonii biofilm formation in a FimA-dependent manner. FimA total protein and cell surface expression were increased upon exposure to CSE whereas Mfa1 was unaffected. CSE exposure did not induce P. gingivalis auto-aggregation but did promote dual species biofilm formation, monitored by microcolony numbers and depth (both, p<0.05). Interestingly, P. gingivalis biofilms grown in the presence of CSE exhibited a lower pro-inflammatory capacity (TNF-α, IL-6) than control biofilms (both, p<0.01). CSE-exposed P. gingivalis bound more strongly to immobilized rGAPDH, the cognate FimA ligand on S. gordonii, than control biofilms (p<0.001) and did so in a dose-dependent manner. Nevertheless, a peptide representing the Mfa1 binding site on S. gordonii, SspB, completely inhibited dual species biofilm formation. Thus, CSE likely augments P. gingivalis biofilm formation by increasing FimA avidity which, in turn, supports initial interspecies interactions and promotes subsequent high affinity Mfa1-SspB interactions driving biofilm growth. CSE induction of P. gingivalis biofilms of limited pro-inflammatory potential may explain the increased persistence of this pathogen in smokers. These findings may also be relevant to other biofilm-induced infectious diseases and conditions

RecA and RadA Proteins of Brucella abortus Do Not Perform Overlapping Protective DNA Repair Functions following Oxidative Burst

Very little is known about the role of DNA repair networks in Brucella abortus and its role in pathogenesis. We investigated the roles of RecA protein, DNA repair, and SOS regulation in B. abortus. While recA mutants in most bacterial species are hypersensitive to UV damage, surprisingly a B. abortus recA null mutant conferred only modest sensitivity. We considered the presence of a second RecA protein to account for this modest UV sensitivity. Analyses of the Brucella spp. genomes and our molecular studies documented the presence of only one recA gene, suggesting a RecA-independent repair process. Searches of the available Brucella genomes revealed some homology between RecA and RadA, a protein implicated in E. coli DNA repair. We considered the possibility that B. abortus RadA might be compensating for the loss of RecA by promoting similar repair activities. We present functional analyses that demonstrated that B. abortus RadA complements a radA defect in E. coli but could not act in place of the B. abortus RecA. We show that RecA but not RadA was required for survival in macrophages. We also discovered that recA was expressed at high constitutive levels, due to constitutive LexA cleavage by RecA, with little induction following DNA damage. Higher basal levels of RecA and its SOS-regulated gene products might protect against DNA damage experienced following the oxidative burst within macrophages. Originally published Journal of Bacteriology, Vol. 188, No. 14, July 200

Evolution of a Membrane Protein Regulon in Saccharomyces

Expression variation is widespread between species. The ability to distinguish regulatory change driven by natural selection from the consequences of neutral drift remains a major challenge in comparative genomics. In this work, we used observations of mRNA expression and promoter sequence to analyze signatures of selection on groups of functionally related genes in Saccharomycete yeasts. In a survey of gene regulons with expression divergence between Saccharomyces cerevisiae and S. paradoxus, we found that most were subject to variation in trans-regulatory factors that provided no evidence against a neutral model. However, we identified one regulon of membrane protein genes controlled by unlinked cis- and trans-acting determinants with coherent effects on gene expression, consistent with a history of directional, nonneutral evolution. For this membrane protein group, S. paradoxus alleles at regulatory loci were associated with elevated expression and altered stress responsiveness relative to other yeasts. In a phylogenetic comparison of promoter sequences of the membrane protein genes between species, the S. paradoxus lineage was distinguished by a short branch length, indicative of strong selective constraint. Likewise, sequence variants within the S. paradoxus population, but not across strains of other yeasts, were skewed toward low frequencies in promoters of genes in the membrane protein regulon, again reflecting strong purifying selection. Our results support a model in which a distinct expression program for the membrane protein genes in S. paradoxus has been preferentially maintained by negative selection as the result of an increased importance to organismal fitness. These findings illustrate the power of integrating expression- and sequence-based tests of natural selection in the study of evolutionary forces that underlie regulatory change

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants

Genomotyping of Coxiella burnetii Using Microarrays Reveals a Conserved Genomotype for Hard Tick Isolates

C. burnetii is a Gram-negative intracellular Y-proteobacteria that causes the zoonotic disease Q fever. Q fever can manifest as an acute or chronic illness. Different typing methods have been previously developed to classify C. burnetii isolates to explore its pathogenicity. Here, we report a comprehensive genomotyping method based on the presence or absence of genes using microarrays. The genomotyping method was then tested in 52 isolates obtained from different geographic areas, different hosts and patients with different clinical manifestations. The analysis revealed the presence of 10 genomotypes organized into 3 groups, with a topology congruent with that obtained through multi-spacer typing. We also found that only 4 genomotypes were specifically associated with acute Q fever, whereas all of the genomotypes could be associated to chronic human infection. Serendipitously, the genomotyping results revealed that all hard tick isolates, including the Nine Mile strain, belong to the same genomotype

In-Vitro Activity of Polymyxin B, Rifampicin, Tigecycline Alone and in Combination against Carbapenem-Resistant Acinetobacter baumannii in Singapore

OBJECTIVE: Carbapenem-resistant Acinetobacter baumannii (CR-AB) is an emerging cause of nosocomial infections worldwide. Combination therapy may be the only viable option until new antibiotics become available. The objective of this study is to identify potential antimicrobial combinations against CR-AB isolated from our local hospitals. METHODS: AB isolates from all public hospitals in Singapore were systematically collected between 2006 and 2007. MICs were determined according to CLSI guidelines. All CR-AB isolates were genotyped using a PCR-based method. Clonal relationship was elucidated. Time-kill studies (TKS) were conducted with polymyxin B, rifampicin and tigecycline alone and in combination using clinically relevant (achievable) unbound concentrations. RESULTS: 31 CR AB isolates were identified. They are multidrug-resistant, but are susceptible to polymyxin B. From clonal typing, 8 clonal groups were identified and 11 isolates exhibited clonal diversity. In single TKS, polymyxin B, rifampicin and tigecycline alone did not exhibit bactericidal activity at 24 hours. In combination TKS, polymyxin plus rifampicin, polymyxin B plus tigecycline and tigecycline plus rifampicin exhibited bactericidal killing in 13/31, 9/31 and 7/31 isolates respectively at 24 hours. Within a clonal group, there may be no consensus with the types of antibiotics combinations that could still kill effectively. CONCLUSION: Monotherapy with polymyxin B may not be adequate against polymyxin B susceptible AB isolates. These findings demonstrate that in-vitro synergy of antibiotic combinations in CR AB may be strain dependant. It may guide us in choosing a pre-emptive therapy for CR AB infections and warrants further investigations