VapC toxin inhibition as a method for prevention of the formation of resting forms of mycobacteria

It has been shown that inactivation of the VapC toxin from the VapВС toxin-antitoxin system prevents mycobacterial cells from transitioning to an ovoid state that meets the criteria of dormancy. The results indicate a potential target for medicines that prevent the development of latent tuberculosis infection and provide a basis to obtain bacterial cells for the testing of compounds that are active towards dormant forms of mycobacteria.     

Rickettsia symbiont in the pea aphid Acyrthosiphon pisum: novel cellular tropism, effect on host fitness, and interaction with the essential symbiont Buchnera

In natural populations of the pea aphid Acyrthosiphon pisum, a facultative bacterial symbiont of the genus Rickettsia has been detected at considerable infection frequencies worldwide. We investigated the effects of the Rickettsia symbiont on the host aphid and also on the coexisting essential symbiont Buchnera. In situ hybridization revealed that the Rickettsia symbiont was specifically localized in two types of host cells specialized for endosymbiosis: secondary mycetocytes and sheath cells. Electron microscopy identified bacterial rods, about 2 mum long and 0.5 mum thick, in sheath cells of Rickettsia-infected aphids. Virus-like particles were sometimes observed in association with the bacterial cells. By an antibiotic treatment, we generated Rickettsia-infected and Rickettsia-eliminated aphid strains with an identical genetic background. Comparison of these strains revealed that Rickettsia infection negatively affected some components of the host fitness. Quantitative PCR analysis of the bacterial population dynamics identified a remarkable interaction between the coexisting symbionts: Buchnera population was significantly suppressed in the presence of Rickettsia, particularly at the young adult stage, when the aphid most actively reproduces. On the basis of these results, we discussed the possible mechanisms that enable the prevalence of Rickettsia infection in natural host populations in spite of the negative fitness effects observed in the laboratory.     

Analysis of Non-Typeable Haemophilous influenzae VapC1 Mutations Reveals Structural Features Required for Toxicity and Flexibility in the Active Site

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.     

Bacteria in ovarioles of females from maleless families of ladybird beetles Adalia bipunctata L. (Coleoptera: Coccinellidae) naturally infected with Rickettsia,Wolbachia, and Spiroplasma

Ovarioles were found to be infected with Spiroplasma, Wolbachia, and Rickettsia in Adalia bipunctata females with maleless progeny in different natural populations. Ooplasm was infected with few Wolbachia bacteria. In ooplasm infected by Rickettsia, bacteria were present in small foci. Spiroplasmas were found encapsulated into ooplasm from the wider intercellular spaces between epithelial and oocyte cells. The cytoplasm of follicular epithelia infected with Rickettsia was heavily destroyed, but the nucleus was intact and free from bacteria. The essential feature of follicular epithelium cells from Spiroplasma and Wolbachia infected A. bipunctata females was inclusions of three types: crystalline, filaments, and concentric myelin-like lamellae. Observations of smears prepared from ovaries of A. bipunctata from natural populations revealed a low concentration of bacteria within a microscopy field (less 10 bacteria) in more than 90% of specimens, and only a few ovaries were heavily infected. Two different ways of bacterial invasion of the oocyte are suggested: Spiroplasma-like, through the intercellular spaces in the epithelium and Rickettsia-like, through the cytoplasm of follicular epithelium cells. Bacteria were not found in germarium zones and we suggest that each follicle is infected from haemolymph.     

细菌毒素-抗毒素系统在噬菌体流产感染中的作用北大核心CSCD

细菌常受到数量众多的噬菌体感染,宿主细菌在和噬菌体竞赛中进化出多样化的分子策略,流产感染(abortive infection,Abi)是其中之一。毒素-抗毒素系统(toxin-antitoxin system,TA)会在细菌受到压力胁迫时表达并介导细菌的低代谢甚至休眠,还能直接减少子代噬菌体形成。此外,部分毒素序列和结构与Cas蛋白高度同源,噬菌体甚至会编码抗毒素类似物来阻遏对应毒素的活性。这表明流产感染中细菌死亡过程导致的噬菌体感染失败与TA功能高度重合,TA可能是噬菌体侵染宿主的主要阻力和防御力量之一。文中基于TA系统的分类和功能,对参与噬菌体流产感染的TA系统进行了综述,并预测具有流产功能的TA系统和其在抗生素开发和疾病治疗中的应用前景。这有助于认识细菌-噬菌体相互作用,并指导噬菌体治疗和合成生物学。     

Obligate intracellular bacteria diversity in unfed Leptotrombidium scutellare larvae highlights novel bacterial endosymbionts of mites

It is well known that the mite Leptotrombidium scutellare carries the pathogen of scrub typhus, Orientia tsutsugamushi. However, our understanding of other bacterial endosymbionts of mites is limited. This study investigated the diversity of the obligate intracellular bacteria carried by L. scutellare using 16S rRNA gene amplicon analysis with next-generation sequencing. The results showed that the detected bacteria were classified into the genera Rickettsia, Wolbachia, and Rickettsiella and an unknown genus of the order Rickettsiales. For further classification of the detected bacteria, a representative read that was most closely related to the assigned taxonomic classification was subjected to homology search and phylogenic analysis. The results showed that some bacteria of the genus Rickettsia were identical or very close to the human pathogens Rickettsia akari, Rickettsia aeschlimannii, Rickettsia felis, and Rickettsia australis. The genetic distance between the genus Wolbachia bacteria in the present study and in previous reports is highly indicative that the bacteria in the present study can be classified as a new taxon of Wolbachia. This study detected obligate intracellular bacteria from unfed mites; thus, the mites did not acquire bacteria from infected animals or any other infectious sources. Finally, the present study demonstrated that various and novel bacterial endosymbionts of mites, in addition to O. tsutsugamushi, might uniquely evolve with the host mites throughout overlapping generations of the mite life cycle. The roles of the bacteria in mites and their pathogenicity should be further examined in studies based on bacterial isolation.     

Axe-Txe,a broad-spectrum proteic toxin-antitoxin system specified by a multidrug-resistant,clinical isolate of Enterococcus faecium

Enterococcal species of bacteria are now acknowledged as leading causes of bacteraemia and other serious nosocomial infections. However, surprisingly little is known about the molecular mechanisms that promote the segregational stability of antibiotic resistance and other plasmids in these bacteria. Plasmid pRUM (24 873 bp) is a multidrug resistance plasmid identified in a clinical isolate of Enterococcus faecium. A novel proteic-based toxin-antitoxin cassette identified on pRUM was demonstrated to be a functional segregational stability module in both its native host and evolutionarily diverse bacterial species. Induced expression of the toxin protein (Txe) of this system resulted in growth inhibition in Escherichia coli. The toxic effect of Txe was alleviated by co-expression of the antitoxin protein, Axe. Homologues of the axe and txe genes are present in the genomes of a diversity of Eubacteria. These homologues (yefM-yoeB) present in the E. coli chromosome function as a toxin-antitoxin mechanism, although the Axe and YefM antitoxin components demonstrate specificity for their cognate toxin proteins in vivo. Axe-Txe is one of the first functional proteic toxin-antitoxin systems to be accurately described for Gram-positive bacteria.     

Determination of ribonuclease sequence-specificity using Pentaprobes and mass spectrometry

The VapBC toxin-antitoxin (TA) family is the largest of nine identified TA families. The toxin, VapC, is a metal-dependent ribonuclease that is inhibited by its cognate antitoxin, VapB. Although the VapBCs are the largest TA family, little is known about their biological roles. Here we describe a new general method for the overexpression and purification of toxic VapC proteins and subsequent determination of their RNase sequence-specificity. Functional VapC was isolated by expression of the nontoxic VapBC complex, followed by removal of the labile antitoxin (VapB) using limited trypsin digestion. We have then developed a sensitive and robust method for determining VapC ribonuclease sequence-specificity. This technique employs the use of Pentaprobes as substrates for VapC. These are RNA sequences encoding every combination of five bases. We combine the RNase reaction with MALDI-TOF MS to detect and analyze the cleavage products and thus determine the RNA cut sites. Successful MALDI-TOF MS analysis of RNA fragments is acutely dependent on sample preparation methods. The sequence-specificity of four VapC proteins from two different organisms (VapC(PAE0151) and VapC(PAE2754) from Pyrobaculum aerophilum, and VapC(Rv0065) and VapC(Rv0617) from Mycobacterium tuberculosis) was successfully determined using the described strategy. This rapid and sensitive method can be applied to determine the sequence-specificity of VapC ribonucleases along with other RNA interferases (such as MazF) from a range of organisms.     

Characterization of a novel toxin-antitoxin module, VapBC, encoded by Leptospira interrogans chromosome

Comparative genomic analysis of the coding sequences (CDSs) of Leptospira interrogans revealed a pair of closely linked genes homologous to the vapBC loci of many other bacteria with respect to both deduced amino acid sequencesand operon organizations. Expression of single vapC gene in Escherichia coli resulted in inhibition of bacterial growth,whereas co-expression of vapBC restored the growth effectively. This phenotype is typical for three other character-ized toxin-antitoxin systems of bacteria, i.e., mazEF[1], reIBE[2] and chplK[3]. The VapC proteins of bacteria and a thermophilic archeae, Solfolobus tokodaii, form a structurally distinguished group of toxin different from the other known toxins of bacteria. Phylogenetic analysis of both toxins and antitoxins of all categories indicated that althought oxins were evolved from divergent sources and may or may not follow their speciation paths (as indicated by their 16s RNA seouences）, co-evolution with their antitoxins was obvious.     

Eicosanoids rescue Spodoptera exigua infected with Xenorhabdus nematophilus, the symbiotic bacteria to the entomopathogenic nematode Steinernema carpocapsae

Xenorhabdus nematophilus is a pathogenic bacterium causing insect haemolymph septicemia, which leads to host insect death. To address the fundamental mechanisms underlying this haemolymph septicemia, or the immunodepressive response of the host insects following bacterial infection, we tested a hypothesis that the insect immune-mediating eicosanoid pathway is blocked by inhibitory action of the bacterium. Haemocoelic injection of the bacteria into the fifth instar larvae of Spodoptera exigua reduced the total number of living haemocytes with postinjection time and resulted in host death in 16 h at 25 degrees C. The lethal efficacy, described by the median lethal bacterial dose (LD(50)), was estimated as 33 colony-forming units per fifth instar larva of S. exigua. The lethal effect of the bacteria on the infected larvae decreased significantly with the addition of exogenous arachidonic acid (10 μg), a precursor of eicosanoids. In comparison, injections of dexamethasone (10 μg), a specific inhibitor of phospholipase A(2), and other eicosanoid biosynthesis inhibitors elevated significantly the bacterial pathogenicity. Live X. nematophilus induced the infected larvae to form less nodules than did the heat-killed bacteria, but the addition of arachidonic acid increased the number of nodules formed significantly in response to live bacterial injection. The treatment with dexamethasone and other inhibitors, however, decreased the nodule formation after injection of heat-killed bacteria. These results indicate that eicosanoids play a role in the immune response of S. exigua, and suggest strongly that X. nematophilus inhibits its eicosanoid pathway, which then results in immunodepressive haemolymph septicemia.     

Structural and functional studies of the Mycobacterium tuberculosis VapBC30 toxin-antitoxin system: implications for the design of novel antimicrobial peptides

Toxin-antitoxin (TA) systems play important roles in bacterial physiology, such as multidrug tolerance, biofilm formation, and arrest of cellular growth under stress conditions. To develop novel antimicrobial agents against tuberculosis, we focused on VapBC systems, which encompass more than half of TA systems in Mycobacterium tuberculosis. Here, we report that theMycobacterium tuberculosis VapC30 toxin regulates cellular growth through both magnesium and manganese ion-dependent ribonuclease activity and is inhibited by the cognate VapB30 antitoxin. We also determined the 2.7-Å resolution crystal structure of the M. tuberculosis VapBC30 complex, which revealed a novel process of inactivation of the VapC30 toxin via swapped blocking by the VapB30 antitoxin. Our study on M. tuberculosis VapBC30 leads us to design two kinds of VapB30 and VapC30-based novel peptides which successfully disrupt the toxin-antitoxin complex and thus activate the ribonuclease activity of the VapC30 toxin. Our discovery herein possibly paves the way to treat tuberculosis for next generation.     

New rapid and simple methods for detection of bacteria and determination of their antibiotic susceptibility by using phage mutants

Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria ("infectious centers"). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.     

Horizontal transmission of the insect symbiont Rickettsia is plant-mediated

Bacteria in the genus Rickettsia, best known as vertebrate pathogens vectored by blood-feeding arthropods, can also be found in phytophagous insects. The presence of closely related bacterial symbionts in evolutionarily distant arthropod hosts presupposes a means of horizontal transmission, but no mechanism for this transmission has been described. Using a combination of experiments with live insects, molecular analyses and microscopy, we found that Rickettsia were transferred from an insect host (the whitefly Bemisia tabaci) to a plant, moved inside the phloem, and could be acquired by other whiteflies. In one experiment, Rickettsia was transferred from the whitefly host to leaves of cotton, basil and black nightshade, where the bacteria were restricted to the phloem cells of the plant. In another experiment, Rickettsia-free adult whiteflies, physically segregated but sharing a cotton leaf with Rickettsia-plus individuals, acquired the Rickettsia at a high rate. Plants can serve as a reservoir for horizontal transmission of Rickettsia, a mechanism which may explain the occurrence of phylogenetically similar symbionts among unrelated phytophagous insect species. This plant-mediated transmission route may also exist in other insect-symbiont systems and, since symbionts may play a critical role in the ecology and evolution of their hosts, serve as an immediate and powerful tool for accelerated evolution.     

New Rapid and Simple Methods for Detection of Bacteria and Determination of Their Antibiotic Susceptibility by Using Phage Mutants

Three new methods applying a novel approach for rapid and simple detection of specific bacteria, based on plaque formation as the end point of the phage lytic cycle, are described. Different procedures were designed to ensure that the resulting plaques were derived only from infected target bacteria (“infectious centers”). (i) A pair of amber mutants that cannot form plaques at concentrations lower than their reversion rate underwent complementation in the tested bacteria; the number of plaques formed was proportional to the concentration of the bacteria that were coinfected by these phage mutants. (ii) UV-irradiated phages were recovered by photoreactivation and/or SOS repair mediated by target bacteria and plated on a recA uvrA bacterial lawn in the dark to avoid recovery of noninfecting phages. (iii) Pairs of temperature-sensitive mutants were allowed to coinfect their target bacteria at the permissive temperature, followed by incubation of the plates at the restrictive temperature to avoid phage infection of the host cells. This method allowed the omission of centrifuging and washing the infected cells. Only phages that recovered by recombination or complementation were able to form plaques. The detection limit was 1 to 10 living Salmonella or Escherichia coli O157 cells after 3 to 5 h. The antibiotic susceptibility of the target bacteria could also be determined in each of these procedures by preincubating the target bacteria with antibiotic prior to phage infection. Bacteria sensitive to the antibiotic lost the ability to form infectious centers.     

Prokaryotic toxin-antitoxin stress response loci

Although toxin-antitoxin gene cassettes were first found in plasmids, recent database mining has shown that these loci are abundant in free-living prokaryotes, including many pathogenic bacteria. For example, Mycobacterium tuberculosis has 38 chromosomal toxin-antitoxin loci, including 3 relBE and 9 mazEF loci. RelE and MazF are toxins that cleave mRNA in response to nutritional stress. RelE cleaves mRNAs that are positioned at the ribosomal A-site, between the second and third nucleotides of the A-site codon. It has been proposed that toxin-antitoxin loci function in bacterial programmed cell death, but evidence now indicates that these loci provide a control mechanism that helps free-living prokaryotes cope with nutritional stress.     

The actin-based motility of the facultative intracellular pathogen Listeria monocytogenes

Summary
The Gram-positive bacterium Listeria monocytogenes is a facultative intracellular parasite that invades and multiplies within diverse eukaryotic cell types. An essential pathogenicity determinant is its ability to move in the host cell cytoplasm and to spread within tissues by directly passing from one cell to another. The propulsive force for intracellular movement is thought to be generated by continuous actin assembly at the rear end of the bacterium. Moving bacteria that reach the plasma membrane induce the formation of long membranous protrusions that are internalized by neighbouring cells, thus mediating the spread of infection. The unrelated pathogens Shigella and Rickettsia use a similar process of actin-based motility to disseminate in infected tissues. This review focuses on the bacterial and cellular factors involved in the actin-based motility of L monocytogenes.