Pleiotropic Action of TGF-Beta in Physiological and Pathological Liver Conditions.

The aim of this study is to review and analyze the pleiotropic effects of TGF-ß in physiological and pathological conditions of the liver, with particular emphasis on its role in immune suppression, wound healing, regulation of cell growth and differentiation, and liver cell apoptosis. A literature review was conducted, including 52 studies, comprising review articles, in vitro and in vivo studies, and meta-analyses. Only studies published in peer-reviewed scientific journals were included in the analysis. TGF-ß is a pleiotropic growth factor that is crucial for the liver, both in physiology and pathophysiology. Although its functions are complex and diverse, TGF-ß plays a constant role in immune suppression, wound healing, and the regulation of cell growth and differentiation. In concentrations exceeding the norm, it can induce the apoptosis of liver cells. Increased TGF-ß levels are observed in many liver diseases, such as fibrosis, inflammation, and steatosis. TGF-ß has been shown to play a key role in many physiological and pathological processes of the liver, and its concentration may be a potential diagnostic and prognostic marker in liver diseases.

Multitier regulation of the E. coli extreme acid stress response by CsrA.

CsrA is an RNA-binding protein that regulates processes critical for growth and survival, including central carbon metabolism, motility, biofilm formation, stress responses, and expression of virulence factors in pathogens. Transcriptomics studies in Escherichia coli suggested that CsrA repressed genes involved in surviving extremely acidic conditions. Here, we examine the effects of disrupting CsrA-dependent regulation on the expression of genes and circuitry for acid stress survival and demonstrate CsrA-mediated repression at multiple levels. We show that this repression is critical for managing the trade-off between growth and survival; overexpression of acid stress genes caused by csrA disruption enhances survival under extreme acidity but is detrimental for growth under mildly acidic conditions. In vitro studies confirmed that CsrA binds specifically to mRNAs of structural and regulatory genes for acid stress survival, causing translational repression. We also found that translation of the top-tier acid stress regulator, evgA, is coupled to that of a small leader peptide, evgL, which is repressed by CsrA. Unlike dedicated acid stress response genes, csrA and its sRNA antagonists, csrB and csrC, did not exhibit a substantial response to acid shock. Furthermore, disruption of CsrA regulation of acid stress genes impacted host-microbe interactions in Caenorhabditis elegans, alleviating GABA deficiencies. This study expands the known regulon of CsrA to genes of the extreme acid stress response of E. coli and highlights a new facet of the global role played by CsrA in balancing the opposing physiological demands of stress resistance with the capacity for growth and modulating host interactions.IMPORTANCETo colonize/infect the mammalian intestinal tract, bacteria must survive exposure to the extreme acidity of the stomach. E. coli does this by expressing proteins that neutralize cytoplasmic acidity and cope with molecular damage caused by low pH. Because of the metabolic cost of these processes, genes for surviving acid stress are tightly regulated. Here, we show that CsrA negatively regulates the cascade of expression responsible for the acid stress response. Increased expression of acid response genes due to csrA disruption improved survival at extremely low pH but inhibited growth under mildly acidic conditions. Our findings define a new layer of regulation in the acid stress response of E. coli and a novel physiological function for CsrA.

Oh my gut! Is the microbial origin of neurodegenerative diseases real?

There is no cure or effective treatment for neurodegenerative protein conformational diseases (PCDs), such as Alzheimer's or Parkinson's diseases, mainly because the etiology of these diseases remains elusive. Recent data suggest that unique changes in the gut microbial composition are associated with these ailments; however, our current understanding of the bacterial role in the pathogenesis of PCDs is hindered by the complexity of the microbial communities associated with specific microbiomes, such as the gut, oral, or vaginal microbiota. The composition of these specific microbiomes is regarded as a unique fingerprint affected by factors such as infections, diet, lifestyle, and antibiotics. All of these factors also affect the severity of neurodegenerative diseases. The majority of studies that reveal microbial contribution are correlational, and various models, including worm, fly, and mouse, are being utilized to decipher the role of individual microbes that may affect disease onset and progression. Recent evidence from across model organisms and humans shows a positive correlation between the presence of gram-negative enteropathogenic bacteria and the pathogenesis of PCDs. While these correlational studies do not provide a mechanistic explanation, they do reveal contributing bacterial species and provide an important basis for further investigation. One of the lurking concerns related to the microbial contribution to PCDs is the increasing prevalence of antibiotic resistance and poor antibiotic stewardship, which ultimately select for proteotoxic bacteria, especially the gram-negative species that are known for intrinsic resistance. In this review, we summarize what is known about individual microbial contribution to PCDs and the potential impact of increasing antimicrobial resistance.

Time-off-pick Assay to Measure Caenorhabditis elegans Motility.

Caenorhabditis elegans is a simple metazoan that is often used as a model organism to study various human ailments with impaired motility phenotypes, including protein conformational diseases. Numerous motility assays that measure neuro-muscular function have been employed using C. elegans . Here, we describe "time-off-pick" (TOP), a novel assay for assessing motility in C. elegans . TOP is conducted by sliding an eyebrow hair under the mid-section of the worm and counting the number of seconds it takes for the worm to crawl completely off. The time it takes for the worm to crawl off the eyebrow hair is proportional to the severity of its motility defect. Other readouts of motility include crawling or swimming phenotypes, and although widely established, have some limitations. For example, worms that are roller mutants are less suitable for crawling or swimming assays. We demonstrated that our novel TOP assay is sensitive to age-dependent changes in motility, thus, providing another more inclusive method to assess motor function in C. elegans . Graphical abstract: Conceptual overview of the "time-off-pick" (TOP) assay. Various C. elegans models exhibit age-dependent defects in motility. The time it takes for a worm to crawl off of an eyebrow pick that is slid under its mid-section is measured in TOP seconds. A greater TOP is indicative of a greater motility defect. Eventually, worms with phenotypes that lead to paralysis will not be able to leave the pick.

Antibiotic prescribing patterns for acute respiratory infections in a free clinic network: A pooled cross-sectional study.

In the United States, overprescribing of antibiotics for viral respiratory infections and antimicrobial resistance continue to be public health concerns. To date, no literature has focused on antibiotic prescribing patterns from free clinics. To address this gap, we used patient-level data from a student-run free clinic network of four primary care clinics to assess factors associated with inappropriate antibiotic prescribing for viral respiratory infections. Treatment plans were deemed inappropriate if any type of antibiotic was prescribed. We used unpaired t-tests and chi-square tests to assess for differences in receiving an inappropriate antibiotic prescription by patient-level factors (i.e., age, race/ethnicity, sex, educational attainment, preferred language, insurance status). Of 298 visits, 22.5% did not meet treatment guidelines. No patient-level factors studied were associated with inappropriate antibiotic prescribing. Our findings suggest other factors, beyond patient-level, may be drivers of variation in antibiotic prescribing in free clinics.

Bacteria-Derived Protein Aggregates Contribute to the Disruption of Host Proteostasis.

Neurodegenerative protein conformational diseases are characterized by the misfolding and aggregation of metastable proteins encoded within the host genome. The host is also home to thousands of proteins encoded within exogenous genomes harbored by bacteria, fungi, and viruses. Yet, their contributions to host protein-folding homeostasis, or proteostasis, remain elusive. Recent studies, including our previous work, suggest that bacterial products contribute to the toxic aggregation of endogenous host proteins. We refer to these products as bacteria-derived protein aggregates (BDPAs). Furthermore, antibiotics were recently associated with an increased risk for neurodegenerative diseases, including Parkinson's disease and amyotrophic lateral sclerosis-possibly by virtue of altering the composition of the human gut microbiota. Other studies have shown a negative correlation between disease progression and antibiotic administration, supporting their protective effect against neurodegenerative diseases. These contradicting studies emphasize the complexity of the human gut microbiota, the gut-brain axis, and the effect of antibiotics. Here, we further our understanding of bacteria's effect on host protein folding using the model Caenorhabditis elegans. We employed genetic and chemical methods to demonstrate that the proteotoxic effect of bacteria on host protein folding correlates with the presence of BDPAs. Furthermore, the abundance and proteotoxicity of BDPAs are influenced by gentamicin, an aminoglycoside antibiotic that induces protein misfolding, and by butyrate, a short-chain fatty acid that we previously found to affect host protein aggregation and the associated toxicity. Collectively, these results increase our understanding of host-bacteria interactions in the context of protein conformational diseases.

Quantification of Bacterial Loads in Caenorhabditis elegans.

Caenorhabditis elegans is a ubiquitous free-living nematode that feeds on bacteria. The organism was introduced into a laboratory setting in the 1970s and has since gained popularity as a model to study host-bacteria interactions. One advantage of using C. elegans is that its intestine can be colonized by the bacteria on which it feeds. Quantifying the bacterial load within C. elegans is an important and easily obtainable metric when investigating host-bacteria interactions. Although quantification of bacteria harbored in C. elegans via whole-worm lysis is not a novel assay, there is great variation between existing methods. To lyse C. elegans, many protocols rely on the use of a hand-held homogenizer, which could introduce systematic error and subsequent variation between researchers performing the same experiment. Here, we describe a method of lysing the intestines of C. elegans to quantify the bacterial load within the intestine. Our method has been optimized for removing exogenous bacteria while maintaining worm paralysis, to ensure no bactericidal agents are swallowed, which could kill bacteria within the intestine and affect results. We utilize and compare the efficiency of two different homogenization tools: a battery-powered hand-held homogenizer, and a benchtop electric homogenizer, where the latter minimizes variability. Thus, our protocol has been optimized to reduce systematic error and decrease the potential for variability among experimenters. Graphic abstract: Simplified overview of the procedure used to quantify the bacterial load within C. elegans. The two different methods are herein described for worm lysis: "Option 1" is a hand-held homogenizer, and "Option 2" is a benchtop homogenizer.

Silver nanoparticles enhance the efficacy of aminoglycosides against antibiotic-resistant bacteria.

As the threat of antimicrobial-resistant bacteria compromises the safety and efficacy of modern healthcare practices, the search for effective treatments is more urgent than ever. For centuries, silver (Ag) has been known to have antibacterial properties and, over the past two decades, Ag-based nanoparticles have gained traction as potential antimicrobials. The antibacterial efficacy of Ag varies with structure, size, and concentration. In the present study, we examined Ag nanoparticles (AgNPs) for their antimicrobial activity and safety. We compared different commercially-available AgNPs against gram-negative Escherichia coli, Pseudomonas aeruginosa, Acinetobacter baumannii, and gram-positive Staphylococcus aureus methicillin-resistant and susceptible strains. The most effective formula of AgNPs tested had single-digit (µg/mL) minimum inhibitory concentrations against gram-negative multidrug-resistant clinical bacterial isolates with novel and emerging mechanisms of resistance. The mode of killing was assessed in E. coli and was found to be bactericidal, which is consistent with previous studies using other AgNP formulations. We evaluated cytotoxicity by measuring physiological readouts using the Caenorhabditis elegans model and found that motility was affected, but not the lifespan. Furthermore, we found that at their antibacterial concentrations, AgNPs were non-cytotoxic to any of the mammalian cell lines tested, including macrophages, stem cells, and epithelial cells. More interestingly, our experiments revealed synergy with clinically relevant antibiotics. We found that a non-toxic and non-effective concentration of AgNPs reduced the minimum inhibitory concentrations of aminoglycoside by approximately 22-fold. Because both aminoglycosides and Ag are known to target the bacterial ribosome, we tested whether Ag could also target eukaryotic ribosomes. We measured the rate of mistranslation at bactericidal concentration and found no effect, indicating that AgNPs are not proteotoxic to the host at the tested concentrations. Collectively, our results suggest that AgNPs could have a promising clinical application as a potential stand-alone therapy or antibiotic adjuvants.

Automating Aggregate Quantification in Caenorhabditis elegans.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

The gigA/gigB Genes Regulate the Growth, Stress Response, and Virulence of Acinetobacter baumannii ATCC 17978 Strain.

Acinetobacter baumannii is an important pathogen of nosocomial infection. Recently, a group of genes, named "gig" (for Growth in Galleria), have been identified in a contemporary multi-drug resistant clinical isolate of A. baumannii-strain AB5075. Among these so-called gig genes, gigA and gigB were found to promote antibiotic resistance, stress survival, and virulence of AB5075 by interacting with the nitrogen phosphotransferase system (PTSNtr). This study aimed to investigate the roles of gigA/gigB, which appear to comprise a stress-signaling pathway (encoding for an atypical two-component system response regulator and a predicted anti-anti-sigma factor, respectively), and the involvement of ptsP (encoding the Enzyme I component of the PTSNtr) in the growth, stress resistance, and virulence of the widely studied A. baumannii strain ATCC 17978. Genetic analyses of strains harboring mutations of gigA and gigB were performed to investigate the roles of these genes in bacterial growth, stress resistance, evading macrophage defense, and killing of Galleria mellonella larva. In contrast with findings from strain AB5075 where gigA and gigB contribute to aminoglycoside resistance, the data presented herein indicate that the loss of gigA/gigB does not impact antibiotic resistance of strain ATCC 17978. Interestingly, however, we found that deletion of gigA/gigB in the ATCC 17978 background imparts a general growth in laboratory medium and also conferred growth and replication defects within murine macrophages and an inability to kill G. mellonella larvae. Importantly, studies as well as the loss of ptsP restored the phenotypes of the gigA/gigB mutant to that of the wild-type. The data presented herein indicate that in A. baumannii ATCC 17978, the gigA/gigB genes play a key role in both growth and virulence traits, but are dispensable for other stress-resistance survival phenotypes, including aminoglycoside resistance. Our findings thus highlight several similarities and also important differences between the gigA/gigB stress-signaling pathway in two commonly studied isolates of this troublesome pathogen.

Yersinia pseudotuberculosis YopJ Limits Macrophage Response by Downregulating COX-2-Mediated Biosynthesis of PGE2 in a MAPK/ERK-Dependent Manner.

Prostaglandin E2 (PGE2) is an essential immunomodulatory lipid released by cells in response to infection with many bacteria, yet its function in macrophage-mediated bacterial clearance is poorly understood. Yersinia overall inhibits the inflammatory circuit, but its effect on PGE2 production is unknown. We hypothesized that one of the Yersinia effector proteins is responsible for the inhibition of PGE2 biosynthesis. We identified that yopB-deficient Y. enterocolitica and Y. pseudotuberculosis deficient in the secretion of virulence proteins via a type 3 secretion system (T3SS) failed to inhibit PGE2 biosynthesis in macrophages. Consistently, COX-2-mediated PGE2 biosynthesis is upregulated in cells treated with heat-killed or T3SS-deficient Y. pseudotuberculosis but diminished in the presence of a MAPK/ERK inhibitor. Mutants expressing catalytically inactive YopJ induce similar levels of PGE2 as heat-killed or ΔyopB Y. pseudotuberculosis, reversed by YopJ complementation. Shotgun proteomics discovered host pathways regulated in a YopJ-mediated manner, including pathways regulating PGE2 synthesis and oxidative phosphorylation. Consequently, this study identified that YopJ-mediated inhibition of MAPK signal transduction serves as a mechanism targeting PGE2, an alternative means of inflammasome inhibition by Yersinia. Finally, we showed that EP4 signaling supports macrophage function in clearing intracellular bacteria. In summary, our unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription, thereby enhancing the intracellular survival of yersiniae. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic molecule to improve the outcomes of specific bacterial infections. Since other pathogens encode YopJ homologs, this mechanism is expected to be present in other infections. IMPORTANCE PGE2 is a critical immunomodulatory lipid, but its role in bacterial infection and pathogen clearance is poorly understood. We previously demonstrated that PGE2 leads to macrophage polarization toward the M1 phenotype and stimulates inflammasome activation in infected macrophages. Finally, we also discovered that PGE2 improved the clearance of Y. enterocolitica. The fact that Y. enterocolitica hampers PGE2 secretion in a type 3 secretion system (T3SS)-dependent manner and because PGE2 appears to assist macrophage in the clearance of this bacterium indicates that targeting of the eicosanoid pathway by Yersinia might be an adaption used to counteract host defenses. Our study identified a mechanism used by Yersinia that obstructs PGE2 biosynthesis in human macrophages. We showed that Y. pseudotuberculosis interferes with PGE2 biosynthesis by using one of its T3SS effectors, YopJ. Specifically, YopJ targets the host COX-2 enzyme responsible for PGE2 biosynthesis, which happens in a MAPK/ER-dependent manner. Moreover, in a shotgun proteomics study, we also discovered other pathways that catalytically active YopJ targets in the infected macrophages. YopJ was revealed to play a role in limiting host LPS responses, including repression of EGR1 and JUN proteins, which control transcriptional activation of proinflammatory cytokine production such as interleukin-1ß. Since YopJ has homologs in other bacterial species, there are likely other pathogens that target and inhibit PGE2 biosynthesis. In summary, our study's unique contribution was to determine a bacterial virulence factor that targets COX-2 transcription. Future studies should investigate whether PGE2 or its stable synthetic derivatives could serve as a potential therapeutic target.

The potential impact of the COVID-19 pandemic on antimicrobial resistance and antibiotic stewardship.

The rapid emergence and spread of antimicrobial resistance continue to kill an estimated 700,000 people annually, and this number is projected to increase ten-fold by 2050. With the lack of data, it is uncertain how the COVID-19 pandemic will affect antimicrobial resistance. Severe disruption of research, innovation, global health programs, and compromised antimicrobial stewardship, infection prevention and control programs, especially in low-and middle-income countries, could affect antimicrobial resistance. However, factors such as strict lockdown, social distancing, vaccination, and the extensive implementation of hand hygiene and face masks, with limited international travel and migration, may also contribute to decreasing AMR. Although the impact of COVID-19 on AMR is global, the adverse effect is likely to be worse in LMICs. In this article, we explore the possible impact of the current pandemic on antibiotic resistance.

Colonization of the Caenorhabditis elegans gut with human enteric bacterial pathogens leads to proteostasis disruption that is rescued by butyrate.

Protein conformational diseases are characterized by misfolding and toxic aggregation of metastable proteins, often culminating in neurodegeneration. Enteric bacteria influence the pathogenesis of neurodegenerative diseases; however, the complexity of the human microbiome hinders our understanding of how individual microbes influence these diseases. Disruption of host protein homeostasis, or proteostasis, affects the onset and progression of these diseases. To investigate the effect of bacteria on host proteostasis, we used Caenorhabditis elegans expressing tissue-specific polyglutamine reporters that detect changes in the protein folding environment. We found that colonization of the C. elegans gut with enteric bacterial pathogens disrupted proteostasis in the intestine, muscle, neurons, and the gonad, while the presence of bacteria that conditionally synthesize butyrate, a molecule previously shown to be beneficial in neurodegenerative disease models, suppressed aggregation and the associated proteotoxicity. Co-colonization with this butyrogenic strain suppressed bacteria-induced protein aggregation, emphasizing the importance of microbial interaction and its impact on host proteostasis. Further experiments demonstrated that the beneficial effect of butyrate depended on the bacteria that colonized the gut and that this protective effect required SKN-1/Nrf2 and DAF-16/FOXO transcription factors. We also found that bacteria-derived protein aggregates contribute to the observed disruption of host proteostasis. Together, these results reveal the significance of enteric infection and gut dysbiosis on the pathogenesis of protein conformational diseases and demonstrate the potential of using butyrate-producing microbes as a preventative and treatment strategy for neurodegenerative disease.

GigC, a LysR Family Transcription Regulator, Is Required for Cysteine Metabolism and Virulence in Acinetobacter baumannii.

A critical facet of mammalian innate immunity involves the hosts' attempts to sequester and/or limit the availability of key metabolic products from pathogens. For example, nutritional immunity encompasses host approaches to limit the availability of key heavy metal ions such as zinc and iron. Previously, we identified several hundred genes in a multidrug-resistant isolate of Acinetobacter baumannii that are required for growth and/or survival in the Galleria mellonella infection model. In the present study, we further characterize one of these genes, a LysR family transcription regulator that we previously named GigC. We show that mutant strains lacking gigC have impaired growth in the absence of the amino acid cysteine and that gigC regulates the expression of several genes involved in the sulfur assimilation and cysteine biosynthetic pathways. We further show that cells harboring a deletion of the gigC gene are attenuated in two murine infection models, suggesting that the GigC protein, likely through its regulation of the cysteine biosynthetic pathway, plays a key role in the virulence of A. baumannii.

Metabolic regulation of gene expression by histone lactylation.

The Warburg effect, which originally described increased production of lactate in cancer, is associated with diverse cellular processes such as angiogenesis, hypoxia, polarization of macrophages and activation of T cells. This phenomenon is intimately linked to several diseases including neoplasia, sepsis and autoimmune diseases1,2. Lactate, which is converted from pyruvate in tumour cells, is widely known as an energy source and metabolic by-product. However, its non-metabolic functions in physiology and disease remain unknown. Here we show that lactate-derived lactylation of histone lysine residues serves as an epigenetic modification that directly stimulates gene transcription from chromatin. We identify 28 lactylation sites on core histones in human and mouse cells. Hypoxia and bacterial challenges induce the production of lactate by glycolysis, and this acts as a precursor that stimulates histone lactylation. Using M1 macrophages that have been exposed to bacteria as a model system, we show that histone lactylation has different temporal dynamics from acetylation. In the late phase of M1 macrophage polarization, increased histone lactylation induces homeostatic genes that are involved in wound healing, including Arg1. Collectively, our results suggest that an endogenous 'lactate clock' in bacterially challenged M1 macrophages turns on gene expression to promote homeostasis. Histone lactylation thus represents an opportunity to improve our understanding of the functions of lactate and its role in diverse pathophysiological conditions, including infection and cancer.

Brucella Periplasmic Protein EipB Is a Molecular Determinant of Cell Envelope Integrity and Virulence.

The Gram-negative cell envelope is a remarkable structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. Multiple molecular systems function to maintain integrity of this essential barrier between the interior of the cell and its surrounding environment. We show that a conserved DUF1849 family protein, EipB, is secreted to the periplasmic space of Brucella species, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual 14-stranded ß-spiral structure that resembles the LolA and LolB lipoprotein delivery system, though the overall fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in Brucella cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of Brucella abortus infection. Transposon disruption of ttpA, which encodes a periplasmic protein containing tetratricopeptide repeats, is synthetically lethal with eipB deletion. ttpA is a reported virulence determinant in Brucella, and our studies of ttpA deletion and overexpression strains provide evidence that this gene also contributes to cell envelope function. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity, which facilitates survival in a mammalian host.IMPORTANCEBrucella species cause brucellosis, a global zoonosis. A gene encoding a conserved DUF1849-family protein, which we have named EipB, is present in all sequenced Brucella and several other genera in the class Alphaproteobacteria The manuscript provides the first functional and structural characterization of a DUF1849 protein. We show that EipB is secreted to the periplasm where it forms a spiral-shaped antiparallel ß protein that is a determinant of cell envelope integrity in vitro and virulence in an animal model of disease. eipB genetically interacts with ttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in select Alphaproteobacteria.

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded ß-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.