Peggy A. Cotter

Caspase-11 Protects Against Bacteria That Escape the Vacuole

Caspase-11–Dependent Pyroptosis Inflammasomes are multiprotein complexes that assemble to initiate immunity to a variety of microorganisms, as well as to sterile tissue injury. Although a role for caspase-1 downstream of inflammasomes is well characterized, the discovery that caspase-1 knockout mice were also deficient in caspase-11 has led to a reassessment of the function of caspase-11. Aachoui et al. (p. 975, published online 24 January; see the Perspective by Cemma and Brumell) now demonstrate that caspase-11 is required for immunity against cytosolic bacteria in mice. Only bacteria that were able to access cytosol-activated caspase-11–dependent pyroptosis, an inflammatory type of cell death. This function of caspase-11 appeared to be independent of canonical inflammasomes. Caspase-11 triggers cell death in response to bacteria that gain access to the cytosol of macrophages. [Also see Perspective by Cemma and Brumell] Caspases are either apoptotic or inflammatory. Among inflammatory caspases, caspase-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only caspase-1 has an established protective role during infection. Here, we report that caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. Although Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) aberrantly enter the cytosol. These mutants triggered caspase-11, which enhanced clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered caspase-11, which protected mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.

Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction

Signal transduction molecules within the two-component family represent a conserved adaptation for the control of genes involved in pathogenesis. The Bordetella virulence control locus, bvgAS, activates and represses gene expression in response to environmental signals. While infection requires virulence gene activation, the role of gene repression during infection is not understood. By altering regulatory genes and reversing regulatory connections, we found evidence that the BvgAS-repressed genes responsible for motility are neither required nor expressed during colonization of the host. Expression of this Bvg- phase-specific phenotype in the Bvg+ growth phase resulted in a defect in tracheal colonization. Therefore, BvgAS promotes virulence both by activating genes required for colonization and by repressing genes that inhibit the development of infection.

A widespread family of polymorphic contact-dependent toxin delivery systems in bacteria

Bacteria have developed mechanisms to communicate and compete with one another in diverse environments. A new form of intercellular communication, contact-dependent growth inhibition (CDI), was discovered recently in Escherichia coli. CDI is mediated by the CdiB/CdiA two-partner secretion (TPS) system. CdiB facilitates secretion of the CdiA ‘exoprotein’ onto the cell surface. An additional small immunity protein (CdiI) protects CDI+ cells from autoinhibition. The mechanisms by which CDI blocks cell growth and by which CdiI counteracts this growth arrest are unknown. Moreover, the existence of CDI activity in other bacteria has not been explored. Here we show that the CDI growth inhibitory activity resides within the carboxy-terminal region of CdiA (CdiA-CT), and that CdiI binds and inactivates cognate CdiA-CT, but not heterologous CdiA-CT. Bioinformatic and experimental analyses show that multiple bacterial species encode functional CDI systems with high sequence variability in the CdiA-CT and CdiI coding regions. CdiA-CT heterogeneity implies that a range of toxic activities are used during CDI. Indeed, CdiA-CTs from uropathogenic E. coli and the plant pathogen Dickeya dadantii have different nuclease activities, each providing a distinct mechanism of growth inhibition. Finally, we show that bacteria lacking the CdiA-CT and CdiI coding regions are unable to compete with isogenic wild-type CDI+ cells both in laboratory media and on a eukaryotic host. Taken together, these results suggest that CDI systems constitute an intricate immunity network with an important function in bacterial competition.

A mutation in the Bordetella bronchiseptica bvgS gene results in reduced virulence and increased resistance to starvation, and identifies a new class of Bvg-regulated antigens.

The Bordetella BvgAS signal‐transduction system has traditionally been viewed as mediating a transition between two distinct phenotypic phases: the Bvg+ phase, characterized by the expression of adhesins and toxins, and the Bvg− phase, characterized by motility in Bordetella bronchiseptica and by the expression of vrg loci in Bordetella pertussis. In B. bronchiseptica, the Bvg+ phase is necessary and sufficient for respiratory tract colonization whereas the Bvg− phase is required for growth under nutrient‐limiting conditions. This report describes the characterization of a mutant that is locked in a Bvg‐intermediate (Bvgi) phase. The mutation conferring this phenotype, designated bvgS‐I1, results in a threonine‐to‐methionine substitution near the primary site of phosphorylation in BvgS. Compared to its Bvg+‐phase‐locked parent, the Bvgi mutant displays increased resistance to nutrient limitation and reduced virulence. Molecular analyses indicate that the mutant has lost the ability to express a subset of Bvg+‐phase factors and has gained the ability to express factors unique to the Bvgi phase. Although identified by mutation, this work indicates that the Bvgi phase is expressed by wild‐type B. bronchiseptica in response to certain (semi‐modulating) environmental conditions. The identification of Bvgi‐specific antigens suggests the existence of a new class of Bvg‐regulated genes. We hypothesize that BvgAS is capable of mediating the expression of a spectrum of phenotypic phases in response to the various environments encountered as Bordetella travels within and between mammalian hosts.

Phosphorelay control of virulence gene expression in Bordetella

In Bordetella, the BvgAS phosphorelay comprising the polydomain sensor, BvgS, and response regulator, BvgA, controls the expression of at least three distinct phenotypic phases in response to differences in the concentration of a few chemical compounds. Each phenotypic phase is characterized by maximal expression of some genes and minimal expression of others, and current data support the hypothesis that each plays a distinct and important role in the Bordetella infectious cycle. The ability to control expression of multiple phenotypic states in response to subtle differences in signal intensity could be a characterizing feature of phosphorelays that contain polydomain sensors.

Type VI secretion: not just for pathogenesis anymore.

Type VI secretion systems (T6SS) have been studied primarily in the context of pathogenic bacteria-host interactions. Recent data suggest, however, that these versatile secretion systems may also function to promote commensal or mutualistic relationships between bacteria and eukaryotes or to mediate cooperative or competitive interactions between bacteria.

Modulation of host immune responses, induction of apoptosis and inhibition of NF‐κB activation by the Bordetella type III secretion system

Bordetella bronchiseptica establishes respiratory tract infections in laboratory animals with high efficiency. Colonization persists for the life of the animal and infection is usually asymptomatic in immunocompetent hosts. We hypothesize that this reflects a balance between immunostimulatory events associated with infection and immunomodulatory events mediated by the bacteria. We have identified 15 loci that are part of a type III secretion apparatus in B. bronchiseptica and three secreted proteins. The functions of the type III secretion system were investigated by comparing the phenotypes of wild‐type bacteria with two strains that are defective in type III secretion using in vivo and in vitro infection models. Type III secretion mutants were defective in long‐term colonization of the trachea in immunocompetent mice. The mutants also elicited higher titres of anti‐Bordetella antibodies upon infection compared with wild‐type bacteria. Type III secretion mutants also showed increased lethal virulence in immunodeficient SCID‐beige mice. These observations suggest that type III‐secreted products of B. bronchiseptica interact with components of both innate and adaptive immune systems of the host. B. bronchiseptica induced apoptosis in macrophages in vitro and inflammatory cells in vivo and type III secretion was required for this process. Infection of an epithelial cell line with high numbers of wild type, but not type III deficient B. bronchiseptica resulted in rapid aggregation of NF‐κB into large complexes in the cytoplasm. NF‐κB aggregation was dependent on type III secretion and aggregated NF‐κB did not respond to TNFα activation, suggesting B. bronchiseptica may modulate host immunity by inactivating NF‐κB. Based on these in vivo and in vitro results, we hypothesize that the Bordetella type III secretion system functions to modulate host immune responses during infection.

Aerobic regulation of cytochrome d oxidase (cydAB ) operon expression in Escherichia coli: roles of Fnr and ArcA in repression and activation

The cydAB operon of Escherichia coli encodes the cytochrome d oxidase complex, one of two aerobic terminal oxidases that catalyses the oxidation of ubiquinol‐8 and the reduction of oxygen to water. This enzyme has a higher affinity for oxygen than the cytochrome o oxidase complex and accumulates as oxygen becomes limiting. Expression of the cydAB operon is microaerobically controlled by the ArcA/ArcB two‐component regulatory system and by Fnr. To understand how ArcA and Fnr contribute to this control, a set of cyd–lacZ reporter fusions were constructed and analysed in vivo. Two cydAB promoters, designated P1 and P2, were identified by primer extension analysis and are located 288 and 173 bp upstream of the start of cydA translation respectively. Transcription from promoter P1 was shown to be regulated by both Fnr and ArcA in response to anaerobiosis. DNaseI footprint experiments revealed the locations of two Fnr binding sites at the P1 promoter: one is centred at the start of cyd transcription, while the other is positioned 53.5 bp upstream. A single ArcA‐phosphate binding site of 49 bp, centred 93 bp upstream of promoter P1, was identified to be sufficient for the activation of cydAB expression. Based on the results of the in vitro and in vivo studies, a working model for ArcA activation and Fnr repression of cydAB transcription is proposed.

Contribution of the fnr and arcA gene products in coordinate regulation of cytochrome o and d oxidase (cyoABCDE and cydAB) genes in Escherichia coli

The individual and the combined effect of the fnr and arcA regulatory gene products on cytochrome o oxidase and cytochrome d oxidase gene expression in Escherichia coli were evaluated using lacZ reporter fusions to the cyo-ABCDE and cydAB operons. Fnr repressed cyo-lacZ and cyd-lacZ expression during anaerobic growth but not during aerobic growth conditions. ArcA functioned as an anaerobic repressor of cyo-lacZ expression while, in contrast, it activated cydAB expression during both aerobic and anaerobic growth. ArcA and Fnr appear to function independently of each other to control cyo-ABCDE operon expression. In contrast, FNR repression of cydAB expression was dependent on arcA+, as indicated by the inability of fnr+ plasmids to repress cyd-lacZ expression in an arcA strain. Under no conditions tested did Fnr activate cydAB expression. Most, but not all, of the observed aerobic/anaerobic regulation of cyo and cyd was accounted for by the two transcriptional regulators. These data suggest the existence of additional levels of anaerobic gene control in E. coli. Additionally, the expression of the fnr regulatory gene, and regulation of the anaerobic respiratory genes, narGHJI, dmsABC and frdABCD, was found to be independent of ArcA.

Bordetella pertussis pathogenesis: current and future challenges

Pertussis, also known as whooping cough, has recently re-emerged as a major public health threat despite high levels of vaccination against the aetiological agent Bordetella pertussis. In this Review, we describe the pathogenesis of this disease, with a focus on recent mechanistic insights into B. pertussis virulence-factor function. We also discuss the changing epidemiology of pertussis and the challenges facing vaccine development. Despite decades of research, many aspects of B. pertussis physiology and pathogenesis remain poorly understood. We highlight knowledge gaps that must be addressed to develop improved vaccines and therapeutic strategies.