Two-Component System Cross-Regulation Integrates Bacillus anthracis Response to Heme and Cell Envelope Stress

Two-component signaling systems (TCSs) are one of the mechanisms that bacteria employ to sense and adapt to changes in the environment. A prototypical TCS functions as a phosphorelay from a membrane-bound sensor histidine kinase (HK) to a cytoplasmic response regulator (RR) that controls target gene expression. Despite significant homology in the signaling domains of HKs and RRs, TCSs are thought to typically function as linear systems with little to no cross-talk between non-cognate HK-RR pairs. Here we have identified several cell envelope acting compounds that stimulate a previously uncharacterized Bacillus anthracis TCS. Furthermore, this TCS cross-signals with the heme sensing TCS HssRS; therefore, we have named it HssRS interfacing TCS (HitRS). HssRS reciprocates cross-talk to HitRS, suggesting a link between heme toxicity and cell envelope stress. The signaling between HssRS and HitRS occurs in the parental B. anthracis strain; therefore, we classify HssRS-HitRS interactions as cross-regulation. Cross-talk between HssRS and HitRS occurs at both HK-RR and post-RR signaling junctions. Finally, HitRS also regulates a previously unstudied ABC transporter implicating this transporter in the response to cell envelope stress. This chemical biology approach to probing TCS signaling provides a new model for understanding how bacterial signaling networks are integrated to enable adaptation to complex environments such as those encountered during colonization of the vertebrate host.     

The Rcs Stress Response and Accessory Envelope Proteins Are Required for De Novo Generation of Cell Shape in Escherichia coli

Interactions with immune responses or exposure to certain antibiotics can remove the peptidoglycan wall of many Gram-negative bacteria. Though the spheroplasts thus created usually lyse, some may survive by resynthesizing their walls and shapes. Normally, bacterial morphology is generated by synthetic complexes directed by FtsZ and MreBCD or their homologues, but whether these classic systems can recreate morphology in the absence of a preexisting template is unknown. To address this question, we treated Escherichia coli with lysozyme to remove the peptidoglycan wall while leaving intact the inner and outer membranes and periplasm. The resulting lysozyme-induced (LI) spheroplasts recovered a rod shape after four to six generations. Recovery proceeded via a series of cell divisions that produced misshapen and branched intermediates before later progeny assumed a normal rod shape. Importantly, mutants defective in mounting the Rcs stress response and those lacking penicillin binding protein 1B (PBP1B) or LpoB could not divide or recover their cell shape but instead enlarged until they lysed. LI spheroplasts from mutants lacking the Lpp lipoprotein or PBP6 produced spherical daughter cells that did not recover a normal rod shape or that did so only after a significant delay. Thus, to regenerate normal morphology de novo, E. coli must supplement the classic FtsZ- and MreBCD-directed cell wall systems with activities that are otherwise dispensable for growth under normal laboratory conditions. The existence of these auxiliary mechanisms implies that they may be required for survival in natural environments, where bacterial walls can be damaged extensively or removed altogether.     

Heme Transfer to the Bacterial Cell Envelope Occurs via a Secreted Hemophore in the Gram-positive Pathogen Bacillus anthracis

To initiate and sustain an infection in mammals, bacterial pathogens must acquire host iron. However, the host''s compartmentalization of large amounts of iron in heme, which is bound primarily by hemoglobin in red blood cells, acts as a barrier to bacterial iron assimilation. Bacillus anthracis, the causative agent of the disease anthrax, secretes two NEAT (near iron transporter) proteins, IsdX1 and IsdX2, which scavenge heme from host hemoglobin and promote growth under low iron conditions. The mechanism of heme transfer from these hemophores to the bacterial cell is not known. We present evidence that the heme-bound form of IsdX1 rapidly and directionally transfers heme to IsdC, a NEAT protein covalently attached to the cell wall, as well as to IsdX2. In both cases, the transfer of heme is mediated by a physical association between the donor and recipient. Unlike Staphylococcus aureus, whose NEAT proteins acquire heme from hemoglobin directly at the bacterial surface, B. anthracis secretes IsdX1 to capture heme in the extracellular milieu and relies on NEAT-NEAT interactions to deliver the bound heme to the envelope via IsdC. Understanding the mechanism of NEAT-mediated iron transport into pathogenic Gram-positive bacteria may provide an avenue for the development of therapeutics to combat infection.     

Cell Envelope Perturbation Induces Oxidative Stress and Changes in Iron Homeostasis in Vibrio cholerae

The Vibrio cholerae type II secretion (T2S) machinery is a multiprotein complex that spans the cell envelope. When the T2S system is inactivated, cholera toxin and other exoproteins accumulate in the periplasmic compartment. Additionally, loss of secretion via the T2S system leads to a reduced growth rate, compromised outer membrane integrity, and induction of the extracytoplasmic stress factor RpoE (A. E. Sikora, S. R. Lybarger, and M. Sandkvist, J. Bacteriol. 189:8484-8495, 2007). In this study, gene expression profiling reveals that inactivation of the T2S system alters the expression of genes encoding cell envelope components and proteins involved in central metabolism, chemotaxis, motility, oxidative stress, and iron storage and acquisition. Consistent with the gene expression data, molecular and biochemical analyses indicate that the T2S mutants suffer from internal oxidative stress and increased levels of intracellular ferrous iron. By using a tolA mutant of V. cholerae that shares a similar compromised membrane phenotype but maintains a functional T2S machinery, we show that the formation of radical oxygen species, induction of oxidative stress, and changes in iron physiology are likely general responses to cell envelope damage and are not unique to T2S mutants. Finally, we demonstrate that disruption of the V. cholerae cell envelope by chemical treatment with polymyxin B similarly results in induction of the RpoE-mediated stress response, increased sensitivity to oxidants, and a change in iron metabolism. We propose that many types of extracytoplasmic stresses, caused either by genetic alterations of outer membrane constituents or by chemical or physical damage to the cell envelope, induce common signaling pathways that ultimately lead to internal oxidative stress and misregulation of iron homeostasis.Vibrio cholerae, a rod-shaped, highly motile, gram-negative bacterium, is the causative agent of the life threatening diarrheal disease cholera (59). The type II secretion (T2S) system plays an important role in the pathogenesis of V. cholerae by secreting cholera toxin (63), which is largely responsible for the symptoms of the disease (33). The T2S system is widespread and well conserved in gram-negative bacteria inhabiting a variety of ecological niches and likely contributes to environmental survival as well as to virulence (11, 21). In V. cholerae, secretion via the T2S machinery is supported by a transenvelope complex of 12 Eps proteins (EpsC to EpsN) and the type 4 prepilin peptidase PilD (VcpD) (25, 44, 63). Transport of exoproteins by the T2S system occurs via a two-step process. The first step, which is either Sec or Tat dependent, requires recognition of the N-terminal signal peptide of the exoproteins and translocation through the inner membrane to the periplasm. Then the folded proteins engage the T2S machinery and are subsequently exported across the outer membrane to the extracellular milieu (23, 29).Besides periplasmic accumulation of exoproteins, additional phenotypes of T2S mutants are reported for an increasing number of species, possibly indicating involvement of the T2S system in other important cellular processes. For example, alterations in outer membrane protein composition have been described for T2S mutants of V. cholerae, Aeromonas hydrophila, marine Vibrio sp. strain 60, and Shewanella oneidensis (30, 32, 63, 64). The levels of outer membrane porins OmpU, OmpT, and OmpS are decreased in T2S mutants of V. cholerae (63, 65), and likewise, disruption of T2S genes in A. hydrophila leads to diminished quantities of OmpF and OmpS (30). Similarly, the amounts of the c-type cytochromes MtrC and OmcA in the outer membranes of S. oneidensis T2S mutants are reduced (64). Furthermore, we have shown that inactivation of the T2S system in V. cholerae results in a reduced growth rate, compromised outer membrane integrity, and, as a consequence, induction of RpoE activity. In addition, our studies showed that V. cholerae T2S mutants are unable to survive the passage through the infant mouse gastrointestinal tract (65). Growth defects at low temperatures under laboratory conditions as well as in tap water and amoebae were also observed for T2S mutants of Legionella pneumophila (68).Interestingly, differential abundance of proteins involved in phosphate metabolism and iron uptake has been revealed by proteomic analysis of culture supernatants isolated from wild-type and T2S mutant strains of Pseudoaltermonas tunicata (22). Based on these results, it has been suggested that the T2S system might be involved in iron acquisition. Similarly, certain T2S mutants of Erwinia chrysanthemi exhibit defects indicative of changes in iron homeostasis (17). It has also been noted that the level of aconitate hydratase, an iron-sulfur cluster-containing enzyme, is reduced in L. pneumophila T2S mutants (16).In this study, in an attempt to explain the phenotypes associated with loss of T2S, we performed microarray gene expression profiling of wild-type and T2S-deficient strains. Our data revealed that inactivation of the T2S machinery results in a metabolic feedback loop leading to oxidative stress and changes in iron metabolism. By analyzing another V. cholerae mutant that shares a similar cell envelope phenotype while remaining competent for T2S, we show that the changes in iron homeostasis and oxidative stress are linked to cell envelope damage and extracytoplasmic stress.     

Evidence that Vpu Modulates HIV-1 Gag-Envelope Interaction towards Envelope Incorporation and Infectivity in a Cell Type Dependent Manner

The HIV-1 Vpu is required for efficient virus particle release from the plasma membrane and intracellular CD4 degradation in infected cells. In the present study, we found that the loss of virus infectivity as a result of envelope (Env) incorporation defect caused by a Gag matrix (MA) mutation (L30E) was significantly alleviated by introducing a start codon mutation in vpu. Inactivation of Vpu partially restored the Env incorporation defect imposed by L30E substitution in MA. This effect was found to be comparable in cell types such as 293T, HeLa, NP2 and GHOST as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). However, in HeLa cells BST-2 knockdown was found to further alleviate the effect of Vpu inactivation on infectivity of L30E mutant. Our data demonstrated that the impaired infectivity of virus particles due to Env incorporation defect caused by MA mutation was modulated by start codon mutation in Vpu.     

Surface Stress Induces a Conserved Cell Wall Stress Response in the Pathogenic Fungus Candida albicans

The human fungal pathogen Candida albicans can grow at temperatures of up to 45°C. Here, we show that at 42°C substantially less biomass was formed than at 37°C. The cells also became more sensitive to wall-perturbing compounds, and the wall chitin levels increased, changes that are indicative of wall stress. Quantitative mass spectrometry of the wall proteome using ¹⁵N metabolically labeled wall proteins as internal standards revealed that at 42°C the levels of the β-glucan transglycosylases Phr1 and Phr2, the predicted chitin transglycosylases Crh11 and Utr2, and the wall maintenance protein Ecm33 increased. Consistent with our previous results for fluconazole stress, this suggests that a wall-remodeling response is mounted to relieve wall stress. Thermal stress as well as different wall and membrane stressors led to an increased phosphorylation of the mitogen-activated protein (MAP) kinase Mkc1, suggesting activation of the cell wall integrity (CWI) pathway. Furthermore, all wall and membrane stresses tested resulted in diminished cell separation. This was accompanied by decreased secretion of the major chitinase Cht3 and the endoglucanase Eng1 into the medium. Consistent with this, cht3 cells showed a similar phenotype. When treated with exogenous chitinase, cell clusters both from stressed cells and mutant strains were dispersed, underlining the importance of Cht3 for cell separation. We propose that surface stresses lead to a conserved cell wall remodeling response that is mainly governed by Mkc1 and is characterized by chitin reinforcement of the wall and the expression of remedial wall remodeling enzymes.     

Extracellular Bacterial Pathogen Induces Host Cell Surface Reorganization to Resist Shear Stress

Bacterial infections targeting the bloodstream lead to a wide array of devastating diseases such as septic shock and meningitis. To study this crucial type of infection, its specific environment needs to be taken into account, in particular the mechanical forces generated by the blood flow. In a previous study using Neisseria meningitidis as a model, we observed that bacterial microcolonies forming on the endothelial cell surface in the vessel lumen are remarkably resistant to mechanical stress. The present study aims to identify the molecular basis of this resistance. N. meningitidis forms aggregates independently of host cells, yet we demonstrate here that cohesive forces involved in these bacterial aggregates are not sufficient to explain the stability of colonies on cell surfaces. Results imply that host cell attributes enhance microcolony cohesion. Microcolonies on the cell surface induce a cellular response consisting of numerous cellular protrusions similar to filopodia that come in close contact with all the bacteria in the microcolony. Consistent with a role of this cellular response, host cell lipid microdomain disruption simultaneously inhibited this response and rendered microcolonies sensitive to blood flow–generated drag forces. We then identified, by a genetic approach, the type IV pili component PilV as a triggering factor of plasma membrane reorganization, and consistently found that microcolonies formed by a pilV mutant are highly sensitive to shear stress. Our study shows that bacteria manipulate host cell functions to reorganize the host cell surface to form filopodia-like structures that enhance the cohesion of the microcolonies and therefore blood vessel colonization under the harsh conditions of the bloodstream.     

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.     

LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.     

A Mathematical Model of the Inflammatory Response to Pathogen Challenge

The human body’s immune response to bacterial challenge, even when successful in controlling the infection, can result in negative consequences for the host, including reduced functionality of associated tissues. We present and analyze a low-dimensional mathematical model of this immune response to pathogen invasion, incorporating the coordinated actions of active immune cells, and both pro- and anti-inflammatory cytokines. The model simulates both the positive (pathogen reduction) and negative (local tissue dysfunction) effects of the immune response and includes the important role of immunologic memory in the process of a return to stasis. This differential equation-based model is sufficiently general to be applicable to a wide range of human tissues and organs.     

The WRKY6 Transcription Factor Modulates PHOSPHATE1 Expression in Response to Low Pi Stress in Arabidopsis

Arabidopsis thaliana WRKY family comprises 74 members and some of them are involved in plant responses to biotic and abiotic stresses. This study demonstrated that WRKY6 is involved in Arabidopsis responses to low-Pi stress through regulating PHOSPHATE1 (PHO1) expression. WRKY6 overexpression lines, similar to the pho1 mutant, were more sensitive to low Pi stress and had lower Pi contents in shoots compared with wild-type seedlings and the wrky6-1 mutant. Immunoprecipitation assays demonstrated that WRKY6 can bind to two W-boxes of the PHO1 promoter. RNA gel blot and β-glucuronidase activity assays showed that PHO1 expression was repressed in WRKY6-overexpressing lines and enhanced in the wrky6-1 mutant. Low Pi treatment reduced WRKY6 binding to the PHO1 promoter, which indicates that PHO1 regulation by WRKY6 is Pi dependent and that low Pi treatment may release inhibition of PHO1 expression. Protein gel blot analysis showed that the decrease in WRKY6 protein induced by low Pi treatment was inhibited by a 26S proteosome inhibitor, MG132, suggesting that low Pi–induced release of PHO1 repression may result from 26S proteosome–mediated proteolysis. In addition, WRKY42 also showed binding to W-boxes of the PHO1 promoter and repressed PHO1 expression. Our results demonstrate that WRKY6 and WRKY42 are involved in Arabidopsis responses to low Pi stress by regulation of PHO1 expression.