Intracellular Niche Research Articles

TolC-Dependent Modulation of Host Cell Death by the Francisella tularensis Live Vaccine Strain

Francisella tularensis is a facultative intracellular, Gram-negative pathogen and the causative agent of tularemia. We previously identified TolC as a virulence factor of the F. tularensis live vaccine strain (LVS) and demonstrated that a ΔtolC mutant exhibits increased cytotoxicity toward host cells and elicits increased proinflammatory responses compared to those of the wild-type (WT) strain. TolC is the outer membrane channel component used by the type I secretion pathway to export toxins and other bacterial virulence factors. Here, we show that the LVS delays activation of the intrinsic apoptotic pathway in a TolC-dependent manner, both during infection of primary macrophages and during organ colonization in mice. The TolC-dependent delay in host cell death is required for F. tularensis to preserve its intracellular replicative niche. We demonstrate that TolC-mediated inhibition of apoptosis is an active process and not due to defects in the structural integrity of the ΔtolC mutant. These findings support a model wherein the immunomodulatory capacity of F. tularensis relies, at least in part, on TolC-secreted effectors. Finally, mice vaccinated with the ΔtolC LVS are protected from lethal challenge and clear challenge doses faster than WT-vaccinated mice, demonstrating that the altered host responses to primary infection with the ΔtolC mutant led to altered adaptive immune responses. Taken together, our data demonstrate that TolC is required for temporal modulation of host cell death during infection by F. tularensis and highlight how shifts in the magnitude and timing of host innate immune responses may lead to dramatic changes in the outcome of infection.

Expression of the Effector Protein IncD in Chlamydia trachomatis Mediates Recruitment of the Lipid Transfer Protein CERT and the Endoplasmic Reticulum-Resident Protein VAPB to the Inclusion Membrane

Chlamydia trachomatis is an obligate intracellular human pathogen responsible for ocular and genital infections. To establish its membrane-bound intracellular niche, the inclusion, C. trachomatis relies on a set of effector proteins that are injected into the host cells or inserted into the inclusion membrane. We previously proposed that insertion of the C. trachomatis effector protein IncD into the inclusion membrane contributes to the recruitment of the lipid transfer protein CERT to the inclusion. Due to the genetically intractable status of C. trachomatis at that time, this model of IncD-CERT interaction was inferred from ectopic expression of IncD and CERT in the host cell. In the present study, we investigated the impact of conditionally expressing a FLAG-tagged version of IncD in C. trachomatis. This genetic approach allowed us to establish that IncD-3×FLAG localized to the inclusion membrane and caused a massive recruitment of the lipid transfer protein CERT that relied on the PH domain of CERT. In addition, we showed that the massive IncD-dependent association of CERT with the inclusion led to an increased recruitment of the endoplasmic reticulum (ER)-resident protein VAPB, and we determined that, at the inclusion, CERT-VAPB interaction relied on the FFAT domain of CERT. Altogether, the data presented here show that expression of the C. trachomatis effector protein IncD mediates the recruitment of the lipid transfer protein CERT and the ER-resident protein VAPB to the inclusion.

Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae

Amoebae serve as hosts for various intracellular bacteria, including human pathogens. These microbes are able to overcome amoebal defense mechanisms and successfully establish a niche for replication, which is usually the cytoplasm. Here, we report on the discovery of a bacterial symbiont that is located inside the nucleus of its Hartmannella sp. host. This symbiont, tentatively named ‘Candidatus Nucleicultrix amoebiphila', is only moderately related to known bacteria (∼90% 16S and 23S rRNA sequence similarity) and member of a novel clade of protist symbionts affiliated with the Rickettsiales and Rhodospirillales. Screening of 16S rRNA amplicon data sets revealed a broad distribution of these bacteria in freshwater and soil habitats. ‘Candidatus Nucleicultrix amoebiphila' traffics within 6 h post infection to the host nucleus. Maximum infection levels are reached after 96–120 h, at which time point the nucleus is pronouncedly enlarged and filled with bacteria. Transmission of the symbionts occurs vertically upon host cell division but may also occur horizontally through host cell lysis. Although we observed no impact on the fitness of the original Hartmannella sp. host, the bacteria are rather lytic for Acanthamoeba castellanii. Intranuclear symbiosis is an exceptional phenomenon, and amoebae represent an ideal model system to further investigate evolution and underlying molecular mechanisms of these unique microbial associations.

A Fluorescent Reporter Reveals On/Off Regulation of the Shigella Type III Secretion Apparatus during Entry and Cell-to-Cell Spread

Numerous pathogenic Gram-negative bacteria use a type three secretion apparatus (T3SA) to translocate effector proteins into host cells. Detecting and monitoring the T3SA of intracellular bacteria within intact host cells has been challenging. Taking advantage of the tight coupling between T3S effector-gene transcription and T3SA activity in Shigella flexneri, together with a fast-maturing green fluorescent protein, we developed reporters to monitor T3SA activity in real time. These reporters reveal a dynamic temporal regulation of the T3SA during the course of infection. T3SA is activated initially during bacterial entry and downregulated subsequently when bacteria gain access to the host cell cytoplasm, allowing replenishment of the bacterial stores of T3S substrates necessary for invading neighboring cells. Reactivation of the T3SA was strictly dependent on actin-based motility and formation of plasma membrane protrusions during cell-to-cell spread. Thus, the T3SA is subject to a tight on/off regulation within the bacterial intracellular niche.

Induction of a Stringent Metabolic Response in Intracellular Stages of Leishmania mexicana Leads to Increased Dependence on Mitochondrial Metabolism

Leishmania parasites alternate between extracellular promastigote stages in the insect vector and an obligate intracellular amastigote stage that proliferates within the phagolysosomal compartment of macrophages in the mammalian host. Most enzymes involved in Leishmania central carbon metabolism are constitutively expressed and stage-specific changes in energy metabolism remain poorly defined. Using 13C-stable isotope resolved metabolomics and 2H2O labelling, we show that amastigote differentiation is associated with reduction in growth rate and induction of a distinct stringent metabolic state. This state is characterized by a global decrease in the uptake and utilization of glucose and amino acids, a reduced secretion of organic acids and increased fatty acid β-oxidation. Isotopomer analysis showed that catabolism of hexose and fatty acids provide C4 dicarboxylic acids (succinate/malate) and acetyl-CoA for the synthesis of glutamate via a compartmentalized mitochondrial tricarboxylic acid (TCA) cycle. In vitro cultivated and intracellular amastigotes are acutely sensitive to inhibitors of mitochondrial aconitase and glutamine synthetase, indicating that these anabolic pathways are essential for intracellular growth and virulence. Lesion-derived amastigotes exhibit a similar metabolism to in vitro differentiated amastigotes, indicating that this stringent response is coupled to differentiation signals rather than exogenous nutrient levels. Induction of a stringent metabolic response may facilitate amastigote survival in a nutrient-poor intracellular niche and underlie the increased dependence of this stage on hexose and mitochondrial metabolism.

Purification of intracellular bacteria: isolation of viable Brucella abortus from host cells.

The pathogenesis of brucellosis depends on the ability of bacteria from the genus Brucella to invade and replicate within animal cells. To understand the molecular pathways used by Brucella spp. to reach its intracellular niche, robust and reproducible bacteria purification protocols that provide enough material for biochemical and molecular biology studies are essential. Here, we describe a detailed methodology designed to extract and purify viable brucellae from mammalian host cells at different time periods of their intracellular cycle. The yield of proteins and nucleic acids is sufficient to perform immunochemical analysis, genetic studies, transcriptomics, and proteomics among others.

Nutrient salvaging and metabolism by the intracellular pathogen Legionella pneumophila

The Gram-negative bacterium Legionella pneumophila is ubiquitous in freshwater environments as a free-swimming organism, resident of biofilms, or parasite of protozoa. If the bacterium is aerosolized and inhaled by a susceptible human host, it can infect alveolar macrophages and cause a severe pneumonia known as Legionnaires' disease. A sophisticated cell differentiation program equips L. pneumophila to persist in both extracellular and intracellular niches. During its life cycle, L. pneumophila alternates between at least two distinct forms: a transmissive form equipped to infect host cells and evade lysosomal degradation, and a replicative form that multiplies within a phagosomal compartment that it has retooled to its advantage. The efficient changeover between transmissive and replicative states is fundamental to L. pneumophila's fitness as an intracellular pathogen. The transmission and replication programs of L. pneumophila are governed by a number of metabolic cues that signal whether conditions are favorable for replication or instead trigger escape from a spent host. Several lines of experimental evidence gathered over the past decade establish strong links between metabolism, cellular differentiation, and virulence of L. pneumophila. Herein, we focus on current knowledge of the metabolic components employed by intracellular L. pneumophila for cell differentiation, nutrient salvaging and utilization of host factors. Specifically, we highlight the metabolic cues that are coupled to bacterial differentiation, nutrient acquisition systems, and the strategies utilized by L. pneumophila to exploit host metabolites for intracellular replication.

Associations between properties linked with persistence in a collection of Staphylococcus aureus isolates from bovine mastitis

Staphylococcus aureus is recognized worldwide as a pathogen causing many serious diseases in humans and animals, and is one of the most important etiological agents of clinical and subclinical bovine mastitis. The aim of the present study was to investigate and correlate properties, that may be associated with persistent mastitis, of S. aureus strains isolated from milk of cows suffering from mastitis: (i) expression of capsular antigens (CP5 or CP8) by specific ELISA; (ii) intracellular survival by invasion of MAC-T cells; and (iii) biofilm production by spectrophotometry analysis after growth in TSBglc. The results showed that (i) the proportion of strains expressing capsular antigen was higher in cap8- than in cap5-positive isolates; (ii) a correlation was observed between the capsular profile and the intracellular survival as well as the biofilm production; and (iii) the capsular profile, biofilm production and intracellular survival were associated with only two agr-groups. Statistical and clustering analysis allowed us to establish different profiles that could be associated with in vivo persistence. Indeed, isolates belonging to agr group II, expressing the capsular antigen CP8 and showing low intracellular survival are probably better adapted to an extracellular niche. Conversely, isolates belonging to agr group I that do not express any capsular antigen (CP5 or CP8) but show high intracellular survival are probably better adapted to an intracellular niche.

Rickettsia Actin-Based Motility Occurs in Distinct Phases Mediated by Different Actin Nucleators

Many intracellular bacterial pathogens undergo actin-based motility to promote cell-cell spread during infection [1]. For each pathogen, motility was assumed to be driven by a single actin polymerization pathway. Curiously, spotted fever group Rickettsia differ from other pathogens in possessing two actin-polymerizing proteins. RickA, an activator of the host Arp2/3 complex, was initially proposed to drive motility [2, 3]. Sca2, a mimic of host formins [4, 5], was later shown to be required for motility [6]. Whether and how their activities are coordinated has remained unclear. Here, we show that each protein directs an independent mode of Rickettsia parkeri motility at different times during infection. Early after invasion, motility is slow and meandering, generating short, curved actin tails that are enriched with Arp2/3 complex and cofilin. Early motility requires RickA and Arp2/3 complex and is correlated with transient RickA localization to the bacterial pole. Later in infection, motility is faster and directionally persistent, resulting in long, straight actin tails. Late motility is independent of Arp2/3 complex and RickA and requires Sca2, which accumulates at the bacterial pole. Both motility pathways facilitate cell-to-cell spread. The ability to exploit two actin assembly pathways may allow Rickettsia to establish an intracellular niche and spread between diverse cells throughout a prolonged infection.

Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages.

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.

New Frontiers in Type III Secretion Biology: the Chlamydia Perspective

Members of the order Chlamydiales comprise a group of exquisitely evolved parasites of eukaryotic hosts that extends from single-celled amoeba to mammals. The most notable are human pathogens and include the agent of oculogenital disease Chlamydia trachomatis, the respiratory pathogen C. pneumoniae, and the zoonotic agent C. psittaci. All of these species are obligate intracellular bacteria that develop within parasitophorous vesicles termed inclusions. This demanding lifestyle necessitates orchestrated entry into nonphagocytic cells, creation of a privileged intracellular niche, and subversion of potent host defenses. All chlamydial genomes contain the coding capacity for a nonflagellar type III secretion system, and this mechanism has arisen as an essential contributor to chlamydial virulence. The emergence of tractable approaches to the genetic manipulation of chlamydiae raises the possibility of explosive progress in understanding this important contributor to chlamydial pathogenesis. This minireview considers challenges and recent advances that have revealed how chlamydiae have maintained conserved aspects of T3S while exploiting diversification to yield a system that exerts a fundamental role in the unique biology of Chlamydia species.

Multiple host kinases contribute to Akt activation during Salmonella infection.

SopB is a type 3 secreted effector with phosphatase activity that Salmonella employs to manipulate host cellular processes, allowing the bacteria to establish their intracellular niche. One important function of SopB is activation of the pro-survival kinase Akt/protein kinase B in the infected host cell. Here, we examine the mechanism of Akt activation by SopB during Salmonella infection. We show that SopB-mediated Akt activation is only partially sensitive to PI3-kinase inhibitors LY294002 and wortmannin in HeLa cells, suggesting that Class I PI3-kinases play only a minor role in this process. However, depletion of PI(3,4) P2/PI(3–5) P3 by expression of the phosphoinositide 3-phosphatase PTEN inhibits Akt activation during Salmonella invasion. Therefore, production of PI(3,4) P2/PI(3–5) P3 appears to be a necessary event for Akt activation by SopB and suggests that non-canonical kinases mediate production of these phosphoinositides during Salmonella infection. We report that Class II PI3-kinase beta isoform, IPMK and other kinases identified from a kinase screen all contribute to Akt activation during Salmonella infection. In addition, the kinases required for SopB-mediated activation of Akt vary depending on the type of infected host cell. Together, our data suggest that Salmonella has evolved to use a single effector, SopB, to manipulate a remarkably large repertoire of host kinases to activate Akt for the purpose of optimizing bacterial replication in its host.

Chlamydia trachomatis homotypic inclusion fusion is promoted by host microtubule trafficking

BackgroundThe developmental cycle of the obligate intracellular pathogen Chlamydia is dependant on the formation of a unique intracellular niche termed the chlamydial inclusion. The inclusion is a membrane bound vacuole derived from host cytoplasmic membrane and is modified significantly by the insertion of chlamydial proteins. A unique property of the inclusion is its propensity for homotypic fusion. The vast majority of cells infected with multiple chlamydial elementary bodies (EBs) contain only a single mature inclusion. The chlamydial protein IncA is required for fusion, however the host process involved are uncharacterized.ResultsHere, through live imaging studies, we determined that the nascent inclusions clustered tightly at the cell microtubule organizing center (MTOC) where they eventually fused to form a single inclusion. We established that factors involved in trafficking were required for efficient fusion as both disruption of the microtubule network and inhibition of microtubule trafficking reduced the efficiency of fusion. Additionally, fusion occurred at multiple sites in the cell and was delayed when the microtubule minus ends were either no longer anchored at a single MTOC or when a cell possessed multiple MTOCs.ConclusionsThe data presented demonstrates that efficient homotypic fusion requires the inclusions to be in close proximity and that this proximity is dependent on chlamydial microtubule trafficking to the minus ends of microtubules.

Bartonella and Brucella--Weapons and Strategies for Stealth Attack

Bartonella spp. and Brucella spp. are closely related α-proteobacterial pathogens that by distinct stealth-attack strategies cause chronic infections in mammals including humans. Human infections manifest by a broad spectrum of clinical symptoms, ranging from mild to fatal disease. Both pathogens establish intracellular replication niches and subvert diverse pathways of the host's immune system. Several virulence factors allow them to adhere to, invade, proliferate, and persist within various host-cell types. In particular, type IV secretion systems (T4SS) represent essential virulence factors that transfer effector proteins tailored to recruit host components and modulate cellular processes to the benefit of the bacterial intruders. This article puts the remarkable features of these two pathogens into perspective, highlighting the mechanisms they use to hijack signaling and trafficking pathways of the host as the basis for their stealthy infection strategies.

Host and bacterial factors that regulate LC3 recruitment to Listeria monocytogenes during the early stages of macrophage infection

Listeria monocytogenes is a bacterial pathogen that can escape the phagosome and replicate in the cytosol of host cells during infection. We previously observed that a population (up to 35%) of L. monocytogenes strain 10403S colocalize with the macroautophagy marker LC3 at 1 h postinfection. This is thought to give rise to spacious Listeria-containing phagosomes (SLAPs), a membrane-bound compartment harboring slow-growing bacteria that is associated with persistent infection. Here, we examined the host and bacterial factors that mediate LC3 recruitment to bacteria at 1 h postinfection. At this early time point, LC3+ bacteria were present within single-membrane phagosomes that are LAMP1+. Protein ubiquitination is known to play a role in targeting cytosolic L. monocytogenes to macroautophagy. However, we found that neither protein ubiquitination nor the ubiquitin-binding adaptor SQSTM1/p62 are associated with LC3+ bacteria at 1 h postinfection. Reactive oxygen species (ROS) production by the CYBB/NOX2 NADPH oxidase was also required for LC3 recruitment to bacteria at 1 h postinfection and for subsequent SLAP formation. Diacylglycerol is an upstream activator of the CYBB/NOX2 NADPH oxidase, and its production by both bacterial and host phospholipases was required for LC3 recruitment to bacteria. Our data suggest that the LC3-associated phagocytosis (LAP) pathway, which is distinct from macroautophagy, targets L. monocytogenes during the early stage of infection within host macrophages and allows establishment of an intracellular niche (SLAPs) associated with persistent infection.

The Host-Encoded Heme Regulated Inhibitor (HRI) Facilitates Virulence-Associated Activities of Bacterial Pathogens

Here we show that cells lacking the heme-regulated inhibitor (HRI) are highly resistant to infection by bacterial pathogens. By examining the infection process in wild-type and HRI null cells, we found that HRI is required for pathogens to execute their virulence-associated cellular activities. Specifically, unlike wild-type cells, HRI null cells infected with the gram-negative bacterial pathogen Yersinia are essentially impervious to the cytoskeleton-damaging effects of the Yop virulence factors. This effect is due to reduced functioning of the Yersinia type 3 secretion (T3S) system which injects virulence factors directly into the host cell cytosol. Reduced T3S activity is also observed in HRI null cells infected with the bacterial pathogen Chlamydia which results in a dramatic reduction in its intracellular proliferation. We go on to show that a HRI-mediated process plays a central role in the cellular infection cycle of the Gram-positive pathogen Listeria . For this pathogen, HRI is required for the post-invasion trafficking of the bacterium to the infected host cytosol. Thus by depriving Listeria of its intracellular niche, there is a highly reduced proliferation of Listeria in HRI null cells. We provide evidence that these infection-associated functions of HRI (an eIF2α kinase) are independent of its activity as a regulator of protein synthesis. This is the first report of a host factor whose absence interferes with the function of T3S secretion and cytosolic access by pathogens and makes HRI an excellent target for inhibitors due to its broad virulence-associated activities.

Chlamydia trachomatis infection results in a modest pro-inflammatory cytokine response and a decrease in T cell chemokine secretion in human polarized endocervical epithelial cells

The endocervical epithelium is a major reservoir for Chlamydia trachomatis in women, and genital infections are extended in their duration. Epithelial cells act as mucosal sentinels by secreting cytokines and chemokines in response to pathogen challenge and infection. We therefore determined the signature cytokine and chemokine response of primary-like endocervix-derived epithelial cells in response to a common genital serovar (D) of C. trachomatis. For these studies, we used a recently-established polarized, immortalized, endocervical epithelial cell model (polA2EN) that maintains, in vitro, the architectural and functional characteristics of endocervical epithelial cells in vivo including the production of pro-inflammatory cytokines. PolA2EN cells were susceptible to C. trachomatis infection, and chlamydiae in these cells underwent a normal developmental cycle as determined by a one-step growth curve. IL1α protein levels were increased in both apical and basolateral secretions of C. trachomatis infected polA2EN cells, but this response did not occur until 72h after infection. Furthermore, protein levels of the pro-inflammatory cytokines and chemokines IL6, TNFα and CXCL8 were not significantly different between C. trachomatis infected polA2EN cells and mock infected cells at any time during the chlamydial developmental cycle up to 120h post-infection. Intriguingly, C. trachomatis infection resulted in a significant decrease in the constitutive secretion of T cell chemokines IP10 and RANTES, and this required a productive C. trachomatis infection. Examination of anti-inflammatory cytokines revealed a high constitutive apical secretion of IL1ra from polA2EN cells that was not significantly modulated by C. trachomatis infection. IL-11 was induced by C. trachomatis, although only from the basolateral membrane. These results suggest that C. trachomatis can use evasion strategies to circumvent a robust pro-inflammatory cytokine and chemokine response. These evasion strategies, together with the inherent immune repertoire of endocervical epithelial cells, may aid chlamydiae in establishing, and possibly sustaining, an intracellular niche in microenvironments of the endocervix in vivo.

The Dot/Icm Effector SdhA Is Necessary for Virulence of Legionella pneumophila in Galleria mellonella and A/J Mice

Legionella pneumophila is an intracellular bacterium that resides within amoebae and macrophages in a specialized compartment termed the Legionella-containing vacuole (LCV). As well as providing an intracellular niche for replication, the LCV helps to prevent the release of bacterial components into the cytoplasm. Recognition of these components as danger signals by the host activates immune responses leading to clearance of the bacterium. Here, we examined the role of two important virulence factors of L. pneumophila, the potent danger signal flagellin and the translocated Dot/Icm type IVB secretion system effector SdhA, which is crucial to maintain LCV integrity, in the Galleria mellonella infection model. We demonstrate that flagellin expression does not contribute to virulence, replication, or induction of clearance mechanisms. Conversely, SdhA expression is important for virulence. We found that in the absence of SdhA, the LCV in hemocytes showed signs of instability and leakage. Furthermore, in contrast to wild-type L. pneumophila, a ΔsdhA mutant caused a transient depletion of hemocytes and reduced mortality. Analysis of the ΔsdhA mutant in the A/J mouse model also showed a significant replication defect. Together, our data underline the crucial importance of SdhA in infection across different model organisms.

The Brucella abortus General Stress Response System Regulates Chronic Mammalian Infection and Is Controlled by Phosphorylation and Proteolysis

Virulence of pathogenic bacteria is often determined by their ability to adapt to stress. The Brucella abortus general stress response (GSR) system is required for chronic mammalian infection and is regulated by phosphorylation and proteolysis. The B. abortus GSR signaling pathway has multiple layers of post-translational control and is a determinant of chronic infection. This study provides new, molecular level insight into chronic Brucella infection. Brucella spp. are adept at establishing a chronic infection in mammals. We demonstrate that core components of the α-proteobacterial general stress response (GSR) system, PhyR and σ(E1), are required for Brucella abortus stress survival in vitro and maintenance of chronic murine infection in vivo. ΔphyR and ΔrpoE1 null mutants exhibit decreased survival under acute oxidative and acid stress but are not defective in infection of primary murine macrophages or in initial colonization of BALB/c mouse spleens. However, ΔphyR and ΔrpoE1 mutants are attenuated in spleens beginning 1 month postinfection. Thus, the B. abortus GSR system is dispensable for colonization but is required to maintain chronic infection. A genome-scale analysis of the B. abortus GSR regulon identified stress response genes previously linked to virulence and genes that affect immunomodulatory components of the cell envelope. These data support a model in which the GSR system affects both stress survival and the interface between B. abortus and the host immune system. We further demonstrate that PhyR proteolysis is a unique feature of GSR control in B. abortus. Proteolysis of PhyR provides a mechanism to avoid spurious PhyR protein interactions that inappropriately activate GSR-dependent transcription. We conclude that the B. abortus GSR system regulates acute stress adaptation and long term survival within a mammalian host and that PhyR proteolysis is a novel regulatory feature in B. abortus that ensures proper control of GSR transcription.

Brucella induces an unfolded protein response via TcpB in macrophages (P3116)

Abstract Brucella melitensis is a facultative intracellular bacterium that invades and replicates within macrophages and dendritic cells. With greater than 500,000 new infections per year, brucellosis is the most prevalent zoonosis worldwide. Currently, a safe and effective human vaccine does not exist and Brucella’s intracellular replicative niche renders the organism resistant to antibiotics. A better understanding of host-pathogen interactions supporting Brucella replication is necessary for vaccine and therapeutic development. Brucella fuses with the endoplasmic reticulum (ER) in order to replicate and may provoke a conserved ER stress response called the Unfolded Protein Response (UPR). B. melitensis infection in RAW 264.7 murine macrophages and bone marrow-derived macrophages upregulated the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing. Upregulation of CHOP and ERdj4 was MyD88-independent whereas XBP1 splicing was MyD88-dependent. A Brucella TcpB mutant showed significantly reduced expression of BiP, CHOP, and ERdj4. Purified TcpB, a microtubule-modulating protein, induced UPR target genes. Also, purified TcpB caused ER restructuring similar to infection with B. melitensis. Finally, Brucella replication was significantly impaired by tauroursodeoxycholic acid, a pharmacologic UPR inhibitor. These results suggest that Brucella induces the UPR to enable its intracellular replication and thus provides a novel therapeutic target for brucellosis treatment.