HMGB1 release from trophoblasts contributes to inflammation during Brucella melitensis infection

Brucella melitensis infection causes acute necrotizing inflammation in pregnant animals; however, the pathophysiological mechanisms leading to placentitis are unknown. Here, we demonstrate that high‐mobility group box 1 (HMGB1) acts as a mediator of placenta inflammation in B. melitensis‐infected pregnant mice model. HMGB1 levels were increased in trophoblasts or placental explant during B. melitensis infection. Inhibition of HMGB1 activity with neutralising antibody significantly reduced the secretion of inflammatory cytokines in B. melitensis‐infected trophoblasts or placenta, whereas administration of recombinant HMGB1 (rHMGB1) increased the inflammatory response. Mechanistically, this decreased inflammatory response results from inhibition of HMGB1 activity, which cause the suppression of both mitogen‐activated protein kinases and nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) activation. Moreover, neutralising antibody to HMGB1 prevented B. melitensis infection‐induced activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in trophoblasts. In contrast, in vitro stimulation of trophoblasts with rHMGB1 caused activation of NADPH oxidase and increased the production of ROS, which contributes to high bacterial burden within trophoblasts or placenta. In vivo, treatment with anti‐HMGB1 antibody increases the number of Brucella survival within placenta in B. melitensis‐infected pregnant mice but successfully reduced the severity of placentitis and abortion.     

Bacterial endosymbiont infections in ‘living fossils’: a case study of North American vaejovid scorpions

Bacterial endosymbionts are common among arthropods, and maternally inherited forms can affect the reproductive and behavioural traits of their arthropod hosts. The prevalence of bacterial endosymbionts and their role in scorpion evolution have rarely been investigated. In this study, 61 samples from 40 species of scorpion in the family Vaejovidae were screened for the presence of the bacterial endosymbionts Cardinium, Rickettsia, Spiroplasma and Wolbachia. No samples were infected by these bacteria. However, one primer pair specifically designed to amplify Rickettsia amplified nontarget genes of other taxa. Similar off‐target amplification using another endosymbiont‐specific primer was also found during preliminary screenings. Results caution against the overreliance on previously published screening primers to detect bacterial endosymbionts in host taxa and suggest that primer specificity may be higher in primers targeting nuclear rather than mitochondrial genes.     

Chemokine‐cleaving Streptococcus pyogenes protease SpyCEP is necessary and sufficient for bacterial dissemination within soft tissues and the respiratory tract

SpyCEP is a Streptococcus pyogenes protease that cleaves CXCL8/IL‐8 and its activity is associated with human invasive disease severity. We investigated the role of SpyCEP in S. pyogenes necrotizing fasciitis and respiratory tract infection in mice using isogenic strains differing only in SpyCEP expression. SpyCEP cleaved human CXCL1, 2, 6 and 8 plus murine CXCL1 and 2 at a structurally conserved site. Mice were infected in thigh muscle with a strain of S. pyogenes that expresses a high level of SpyCEP, or with an isogenic non‐SpyCEP expressing strain. SpyCEP expression by S. pyogenes hindered bacterial clearance from muscle, and enhanced bacterial spread, associated with cleavage of murine chemoattractant CXCL1. Mice were then infected with Lactococcus lactis strains that differed only in SpyCEP expression. In contrast to the parent L. lactis strain (lacks SpyCEP), which was avirulent when administered intramuscularly, infection with a strain that expressed SpyCEP heterologously led to dramatic systemic illness within 24 h, failure to clear bacteria from muscle and marked dissemination to other organs. In the upper airways, SpyCEP expression was required for survival of L. lactis but not S. pyogenes. However, dissemination of S. pyogenes to the lung was SpyCEP‐dependent and was associated with evidence of chemokine cleavage. Taken together, the studies provide clear evidence that SpyCEP is necessary and sufficient for systemic bacterial dissemination from a soft tissue focus in this model and also underlies dissemination in the respiratory tract.     

Pathogen effects on vegetative and floral odours mediate vector attraction and host exposure in a complex pathosystem

Pathogens can alter host phenotypes in ways that influence interactions between hosts and other organisms, including insect disease vectors. Such effects have implications for pathogen transmission, as well as host exposure to secondary pathogens, but are not well studied in natural systems, particularly for plant pathogens. Here, we report that the beetle‐transmitted bacterial pathogen Erwinia tracheiphila – which causes a fatal wilt disease – alters the foliar and floral volatile emissions of its host (wild gourd, Cucurbita pepo ssp. texana) in ways that enhance both vector recruitment to infected plants and subsequent dispersal to healthy plants. Moreover, infection by Zucchini yellow mosaic virus (ZYMV), which also occurs at our study sites, reduces floral volatile emissions in a manner that discourages beetle recruitment and therefore likely reduces the exposure of virus‐infected plants to the lethal bacterial pathogen – a finding consistent with our previous observation of dramatically reduced wilt disease incidence in ZYMV‐infected plants.     

Autophagy and the cytoskeleton

Actin-based motility is used by various pathogens such as Listeria and Shigella for dissemination within cells

and tissues, yet host factors counteracting this process have not been identified. We have recently discovered that infected host cells can prevent actin-based motility of Shigella by compartmentalizing bacteria inside ‘septin cages,’ revealing a novel mechanism of host defense that restricts dissemination. Because bacterial proteins controlling actin-based motility also regulate the autophagy process, we hypothesized and then established a link between septin caging and autophagy. Together, these results unveiled the first cellular mechanism that counteracts pathogen dissemination. Understanding the role of septins, a so far poorly characterized component of the cytoskeleton, will thus provide new insights into bacterial infection and autophagy.     

Identification of the essential Brucella melitensis porin Omp2b as a suppressor of Bax-induced cell death in yeast in a genome-wide screening

Background

Inhibition of apoptosis is one of the mechanisms selected by numerous intracellular pathogenic bacteria to control their host cell. Brucellae, which are the causative agent of a worldwide zoonosis, prevent apoptosis of infected cells, probably to support survival of their replication niche.

Methodology/Principal Findings

In order to identify Brucella melitensis anti-apoptotic effector candidates, we performed a genome-wide functional screening in yeast. The B. melitensis ORFeome was screened to identify inhibitors of Bax-induced cell death in S. cerevisiae. B. melitensis porin Omp2b, here shown to be essential, prevents Bax lethal effect in yeast, unlike its close paralog Omp2a. Our results based on Omp2b size variants characterization suggest that signal peptide processing is required for Omp2b effect in yeast.

Conclusion/Significance

We report here the first application to a bacterial genome-wide library of coding sequences of this “yeast-rescue” screening strategy, previously used to highlight several new apoptosis regulators. Our work provides B. melitensis proteins that are candidates for an anti-apoptotic function, and can be tested in mammalian cells in the future. Hypotheses on possible molecular mechanisms of Bax inhibition by the B. melitensis porin Omp2b are discussed.     

Innate immune recognition of flagellin limits systemic persistence of Brucella

Brucella are facultative intracellular bacteria that cause chronic infections by limiting innate immune recognition. It is currently unknown whether Brucella FliC flagellin, the monomeric subunit of flagellar filament, is sensed by the host during infection. Here, we used two mutants of Brucella melitensis, either lacking or overexpressing flagellin, to show that FliC hinders bacterial replication in vivo. The use of cells and mice genetically deficient for different components of inflammasomes suggested that FliC was a target of the cytosolic innate immune receptor NLRC4 in vivo but not in macrophages in vitro where the response to FliC was nevertheless dependent on the cytosolic adaptor ASC, therefore suggesting a new pathway of cytosolic flagellin sensing. However, our work also suggested that the lack of TLR5 activity of Brucella flagellin and the regulation of its synthesis and/or delivery into host cells are both part of the stealthy strategy of Brucella towards the innate immune system. Nevertheless, as a flagellin‐deficient mutant of B. melitensis wasfound to cause histologically demonstrable injuries in the spleen of infected mice, we suggested that recognition of FliC plays a role in the immunological stand‐off between Brucella and its host, which is characterized by a persistent infection with limited inflammatory pathology.     

Cryptic diversity,reproductive isolation and cytoplasmic incompatibility in a classic biological control success story

Molecular genetics and symbiont diagnostics have revolutionized our understanding of insect species diversity, and the transformative effects of bacterial symbionts on host life history. Encarsia inaron is a parasitoid wasp that has been shown to harbour two bacterial endosymbionts, Wolbachia and Cardinium. Known then as E. partenopea, it was introduced to the USA in the late 1980s from populations collected in Italy and Israel for the biological control of an ornamental tree pest, the ash whitefly, Siphoninus phillyreae. We studied natural populations from sites in the USA, the Mediterranean and the Middle East as well as from a Cardinium‐infected laboratory culture established from Italy, with the aims of characterizing these populations genetically, testing reproductive isolation, determining symbiont infection status in their native and introduced range, and determining symbiont role. The results showed that the two Encarsia populations introduced to the USA are genetically distinct, reproductively isolated, have different symbionts and different host–symbiont interactions, and can be considered different biological species. One (‘E. inaron’) is doubly infected by Wolbachia and Cardinium, while only Cardinium is present in the other (‘E. partenopea’). The Cardinium strains in the two species are distinct, although closely related, and crossing tests indicate that the Cardinium infecting ‘E. partenopea’ induces cytoplasmic incompatibility. The frequency of symbiont infection found in the native and introduced range of these wasps was similar, unlike the pattern seen in some other systems. These results also lead to a retelling of a successful biological control story, with several more characters than had been initially described.     

Complex interactions between bacteria and haemosporidia in coinfected hosts: An experiment

1. Hosts are typically coinfected by multiple parasite species whose interactions might be synergetic or antagonistic, producing unpredictable physiological and pathological impacts on the host. This study shows the interaction between Plasmodium spp. and Leucocytozoon spp. in birds experimentally infected or not infected with Mycoplasma gallisepticum.
2. In 1994, the bacterium Mycoplasma gallisepticum jumped from poultry to wild birds in which it caused a major epidemic in North America. Birds infected with M. gallisepticum show conjunctivitis as well as increased levels of corticosterone.
3. Malaria and other haemosporidia are widespread in birds, and chronic infections become apparent with the detectable presence of the parasite in peripheral blood in response to elevated levels of natural or experimental corticosterone levels.
4. Knowing the immunosuppressive effect of corticosterone on the avian immune system, we tested the hypothesis that chronic infections of Plasmodium spp. and Leucocytozoon spp. in house finches would respond to experimental inoculation with M. gallisepticum as corticosterone levels are known to increase following inoculation.
5. Plasmodium spp. infection intensity increased within days of M. gallisepticum inoculation as shown both by the appearance of infected erythrocytes and by the increase in the number and the intensity of positive PCR tests.
6. Leucocytozoon spp. infection intensity increased when Plasmodium spp. infection intensity increased, but not in response to M. gallisepticum inoculation. Leucocytozoon spp. and Plasmodium spp. seemed to compete in the host as shown by a negative correlation between the changes in their PCR score when both pathogens were present in the same individual.
7. Host responses to coinfection with multiple pathogens measured by the hematocrit and white blood cell count depended on the haemosporidian community composition. Host investment in the leukocyte response was higher in the single‐haemosporidia‐infected groups when birds were infected with M. gallisepticum.
8. A trade‐off was observed between the immune control of the chronic infection (Plasmodium spp./Leucocytozoon spp.) and the immune response to the novel bacterial infection (M. gallisepticum).

     

Vertically transmitted symbiont reduces host fitness along temperature gradient

Parasites with exclusive vertical transmission from host parent to offspring are an evolutionary puzzle. With parasite fitness entirely linked to host reproduction, any fitness cost for infected hosts risks their selective elimination. Environmental conditions likely influence parasite impact and thereby the success of purely vertical transmission strategies. We tested for temperature‐dependent virulence of Caedibacter taeniospiralis, a vertically transmitted bacterial symbiont of the protozoan Paramecium tetraurelia. We compared growth of infected and cured host populations at five temperatures (16–32 °C). Infection reduced host density at all temperatures, with a peak of ?30% at 28 °C. These patterns were largely consistent across five infected Paramecium strains. Similar to Wolbachia symbionts, C. taeniospiralis may compensate fitness costs by conferring to the host a ‘killer trait’, targeting uninfected competitors. Considerable loss of infection at 32 °C suggests that killer efficacy is not universal and that limited heat tolerance restricts the conditions for persistence of C. taeniospiralis.     

SKIP,the Host Target of the Salmonella Virulence Factor SifA,Promotes Kinesin‐1‐Dependent Vacuolar Membrane Exchanges

In Salmonella‐infected cells, the bacterial effector SifA forms a functional complex with the eukaryotic protein SKIP (SifA and kinesin‐interacting protein). The lack of either partner has important consequences on the intracellular fate and on the virulence of this pathogen. In addition to SifA, SKIP binds the microtubule‐based motor kinesin‐1. Yet the absence of SifA or SKIP results in an unusual accumulation of kinesin‐1 on the bacterial vacuolar membrane. To understand this apparent contradiction, we investigated the interaction between SKIP and kinesin‐1 and the function of this complex. We show that the C‐terminal RUN (RPIP8, UNC‐14 and NESCA) domain of SKIP interacted specifically with the tetratricopeptide repeat (TPR) domain of the kinesin light chain. Overexpression of SKIP induced a microtubule‐ and kinesin‐1‐dependent anterograde movement of late endosomal/lysosomal compartments. In infected cells, SifA contributed to the fission of vesicles from the bacterial vacuole and the SifA/SKIP complex was required for the formation and/or the anterograde transport of kinesin‐1‐enriched vesicles. These observations reflect the role of SKIP as a linker and/or an activator for kinesin‐1.     

Host tissues as microhabitats for Wolbachia and quantitative insights into the bacterial community in terrestrial isopods

Animal–bacterial symbioses are highly dynamic in terms of multipartite interactions, both between the host and its symbionts as well as between the different bacteria constituting the symbiotic community. These interactions will be reflected by the titres of the individual bacterial taxa, for example via host regulation of bacterial loads or competition for resources between symbionts. Moreover, different host tissues represent heterogeneous microhabitats for bacteria, meaning that host‐associated bacteria might establish tissue‐specific bacterial communities. Wolbachia are widespread endosymbiotic bacteria, infecting a large number of arthropods and filarial nematodes. However, relatively little is known regarding direct interactions between Wolbachia and other bacteria. This study represents the first quantitative investigation of tissue‐specific Wolbachia–microbiota interactions in the terrestrial isopod Armadillidium vulgare. To this end, we obtained a more complete picture of the Wolbachia distribution patterns across all major host tissues, integrating all three feminizing Wolbachia strains (wVulM, wVulC, wVulP) identified to date in this host. Interestingly, the different Wolbachia strains exhibited strain‐specific tissue distribution patterns, with wVulM reaching lower titres in most tissues. These patterns were consistent across different host genetic backgrounds and might reflect different co‐evolutionary histories between the Wolbachia strains and A. vulgare. Moreover, Wolbachia‐infected females carried higher total bacterial loads in several, but not all, tissues, irrespective of the Wolbachia strain. Taken together, this quantitative approach indicates that Wolbachia is part of a potentially more diverse bacterial community, as exemplified by the presence of highly abundant bacterial taxa in the midgut caeca of several A. vulgare populations.     

Brucella melitensis 16MΔhfq attenuation confers protection against wild‐type challenge in BALB/c mice

Brucellosis is a globally distributed zoonotic disease that causes animal and human diseases. Although effective, the current Brucella vaccines (Rev.1 and M5‐90) have several drawbacks. The first involves residual virulence for animals and humans and the second is the inability to differentiate natural infection from that caused by vaccination. Therefore, Brucella melitensis 16M hfq mutant (16MΔhfq) was constructed to overcome these drawbacks. Similarly to Rev.1 and M5‐90, 16MΔhfq reduces survival in macrophages and mice and induces strong protective immunity in BALB/c mice. Moreover, these vaccines elicit anti‐Brucella‐specific IgG1 and IgG2a subtype responses and induce secretion of gamma interferon and interleukin‐4. The Hfq antigen also allows serological differentiation between infected and vaccinated animals. These results show that 16MΔhfq is an ideal live attenuated vaccine candidate against virulent Brucella melitensis 16M infection. It will be further evaluated in sheep.     

First Report of Curtobacterium flaccumfaciens pv. flaccumfaciens Causing Cowpea Bacterial Wilt in Iran

During surveys in cowpea fields of Marand County, East Azerbaijan province, Iran, in the summer of 2013, a suspected bacterial disease was observed on cowpea leaves as tan spots and interveinal necrotic lesions surrounded by chlorotic margins. The disease was of high incidence where some fields had been fully destroyed and severity of the disease in some fields had reached up to 70%. Gram‐positive, yellow‐pigmented, coryneform bacteria were isolated from infected leaves. Pathogenicity of isolates was confirmed on 20‐day‐old cowpea (cv. Khoy) plants, and they were identified as Curtobacterium flaccumfaciens pv. flaccumfaciens based on biochemical test results confirmed using specific PCR primers. This is the first report of C. flaccumfaciens pv. flaccumfaciens, the causal agent of cowpea bacterial wilt in Iran.     

Mosquitoes host communities of bacteria that are essential for development but vary greatly between local habitats

Mosquitoes are insects of interest because several species vector disease‐causing pathogens to humans and other vertebrates. We previously reported that mosquitoes from long‐term laboratory cultures require living bacteria in their gut to develop, but development does not depend on particular species of bacteria. Here, we focused on three distinct but interrelated areas of study to better understand the role of bacteria in mosquito development by studying field and laboratory populations of Aedes aegypti, Aedes albopictus and Culex quinquefasciatus from the southeastern United States. Sequence analysis of bacterial 16S rRNA gene amplicons showed that bacterial community composition differed substantially in larvae from different collection sites, whereas larvae from the same site shared similarities. Although previously unknown to be infected by Wolbachia, results also indicated that Ae. aegypti from one field site hosted a dual infection. Regardless of collection site or factors like Wolbachia infection, however, each mosquito species required living bacteria in their digestive tract to develop. Results also identified several concerns in using antibiotics to eliminate the bacterial community in larvae in order to study its developmental consequences. Altogether, our results indicate that several mosquito species require living bacteria for development. We also hypothesize these species do not rely on particular bacteria because larvae do not reliably encounter the same bacteria in the aquatic habitats they develop in.     

Host nuclear genotype influences phenotype of a conditional mutualist symbiont

Arthropods commonly carry maternally inherited intracellular bacterial symbionts that may profoundly influence host biology and evolution. The intracellular symbiont Rickettsia sp. nr. bellii swept rapidly into populations of the sweetpotato whitefly Bemisia tabaci in the south‐western USA. Previous laboratory experiments showed female‐bias and fitness benefits were associated with Rickettsia infection, potentially explaining the high frequencies of infection observed in field populations, but the effects varied with whitefly genetic line. Here, we explored whether host extranuclear or nuclear genes influenced the variation in the Rickettsia–host phenotype in two genetic lines of the whitefly host, each with Rickettsia‐infected and uninfected sublines. Introgression between the Rickettsia‐infected subline of one genetic line and the Rickettsia‐uninfected subline of the other was used to create two new sublines, each with the maternally inherited extranuclear genetic lineages of one line (Rickettsia, two other symbionts and the mitochondria) and the nuclear genotype of the other. Performance assays comparing the original and new lines showed that in addition to Rickettsia, the interaction of Rickettsia infection with host nuclear genotype influenced development time and the sex ratio of the progeny, whereas the extranuclear genotype did not. Host nuclear genotype, but not extranuclear genotype, also influenced the titre of Rickettsia. Our results support the hypothesis that differences in host nuclear genotype alone may explain considerable within‐population variation in host–symbiont phenotype and may contribute to the observed variation in Rickettsia–whitefly interactions worldwide.     

The candidate vaccine strain Brucella ovis ∆abcBA is protective against Brucella melitensis infection in mice

Brucellosis is a major zoonotic disease, and Brucella melitensis is the species most often associated with human infection. Vaccination is the most efficient tool for controlling animal brucellosis, with a consequent decrease of incidence of human infections. Commercially available live attenuated vaccines provide some degree of protection, but retain residual pathogenicity to human and animals. In this study, Brucella ovis ∆abcBA (Bo∆abcBA), a live attenuated candidate vaccine strain, was tested in two formulations (encapsulated with alginate and alginate plus vitelline protein B [VpB]) to immunize mice against experimental challenge with B. melitensis strain 16M. One week after infection, livers and spleens of immunized mice had reduced numbers of the challenge strain B. melitensis 16M when compared with those of nonimmunized mice, with a reduction of approximately 1-log₁₀ of B. melitensis 16M count in the spleens from immunized mice. Moreover, splenocytes stimulated with B. melitensis antigens in vitro secreted IFN-γ when mice had been immunized with Bo∆abcBA encapsulated with alginate plus VpB, but not with alginate alone. Body and liver weights were similar among groups, although spleens from mice immunized with Bo∆abcBA encapsulated with alginate were larger than those immunized with Bo∆abcBA encapsulated with alginate plus VpB or nonimmunized mice. This study demonstrated that two vaccine formulations containing Bo∆abcBA protected mice against experimental challenge with B. melitensis.     

Comparison of spleen transcriptomes of two wild rodent species reveals differences in the immune response against Borrelia afzelii

Different host species often differ considerably in susceptibility to a given pathogen, but the causes of such differences are rarely known. The natural hosts of the tick‐transmitted bacterium Borrelia afzelii, which is one of causative agents of Lyme borreliosis in humans, include a variety of small mammals like voles and mice. Previous studies have shown that B. afzelii‐infected bank voles (Myodes glareolus) have about ten times higher bacterial load than infected yellow‐necked mice (Apodemus flavicollis), indicating that these two species differ in resistance. In this study, we compared the immune response to B. afzelii infection in these host species by using RNA sequencing to quantify gene expression in spleen. Gene set enrichment analysis (GSEA) showed that several immune pathways were down‐regulated in infected animals in both bank voles and yellow‐necked mice. Moreover, IFNα response was up‐regulated in B. afzelii‐infected yellow‐necked mice, while IL6 signaling and the complement pathway were down‐regulated in infected bank voles; differences in regulation of these three pathways between bank voles and yellow‐necked mice could thus contribute to the difference in resistance to B. afzelii between the species. This study provides knowledge of gene expression induced by a zoonotic pathogen in its natural host, and possible species‐specific regulation of immune responses associated with resistance.     

Insect eggs induce a systemic acquired resistance in Arabidopsis

Although they constitute an inert stage of the insect's life, eggs trigger plant defences that lead to egg mortality or attraction of egg parasitoids. We recently found that salicylic acid (SA) accumulates in response to oviposition by the Large White butterfly Pieris brassicae, both in local and systemic leaves, and that plants activate a response that is similar to the recognition of pathogen‐associated molecular patterns (PAMPs), which are involved in PAMP‐triggered immunity (PTI). Here we discovered that natural oviposition by P. brassicae or treatment with egg extract inhibit growth of different Pseudomonas syringae strains in Arabidopsis through the activation of a systemic acquired resistance (SAR). This egg‐induced SAR involves the metabolic SAR signal pipecolic acid, depends on ALD1 and FMO1, and is accompanied by a stronger induction of defence genes upon secondary infection. Although P. brassicae larvae showed a reduced performance when feeding on Pseudomonas syringae‐infected plants, this effect was less pronounced when infected plants had been previously oviposited. Altogether, our results indicate that egg‐induced SAR might have evolved as a strategy to prevent the detrimental effect of bacterial pathogens on feeding larvae.