Interaction with a lipid membrane: a key step in bacterial toxins virulence

Bacterial toxins are secreted as soluble proteins. However, they have to interact with a cell lipid membrane either to permeabilize the cells (pore forming toxins) or to enter into the cytosol to express their enzymatic activity (translocation toxins). The aim of this review is to suggest that the strategies developed by toxins to insert in a lipid membrane is mediated by their structure. Two categories, which contains both pore forming and translocation toxins, are emerging: alpha helical proteins containing hydrophobic domains and beta sheets proteins in which no hydrophobicity can be clearly detected. The first category would rather interact with the membrane through multi-spanning helical domains whereas the second category would form a beta barrel in the membrane.     

Identification of M cells and their distribution in rabbit intestinal Peyer's patches and appendix

The distribution of intestinal membranous (M) cells has been studied within the follicle-associated epithelium of rabbit Peyer's patches and appendix. Vimentin expression has been assessed as a primary criterion to identify rabbit M cells in tissue sections and in whole tissue preparations. This criterion has been compared to the use of the absence of alkaline phosphatase which, due to its heterogeneous distribution within the enterocyte population, is less reliable than vimentin expression as a marker for rabbit M cells. The pattern of vimentin immunostaining revealed that the majority of M cells are located in the periphery of the follicle-associated epithelium, the dome apex being largely free of M cells. This distribution was confirmed by scanning electron microscopy. Vimentin is also expressed by follicle-associated epithelial cells in the vicinity of crypts which lack the typical lymphocyte-containing pocket of M cells. Cytoplasmic peanut agglutinin binding coincides with vimentin-expression throughout the follicle-associated epithelium but is absent from vimentin-negative enterocytes. The co-localisation of these two phenotypic markers in both M cells and epithelial cells adjacent to crypts, which lack the typical morphology of fully developed rabbit M cells, suggests that they correspond to immature M cells which by their location appear to derive directly from undifferentiated crypt stem cells and not from mature columnar enterocytes.     

Interaction of rabbit lymph node cells with bacteria (Salmonella paratyphi B)

In five rabbits immunized withSalmonella paratyphi B antigen in all four paws, incubation of the regional lymph node cells with the same bacteria was followed by a significant shift of some of the bacteria from the supernatant of the culture medium to the sediment (as compared with the controls). In six rabbits, bactericidal activity was demonstrated in extracts prepared from the regional lymph nodes only 30 hours after immunization. No antibodies were present in extracts from control rabbits up to the fifth day after immunization.     

Highly processive motility is not a general feature of the kinesins

Evidence is presented that the kinesin-related ncd protein is not as processive as kinesin. In low surface density motility experiments, a dimeric ncd fusion protein behaved mechanistically more similar to non-processive myosins than to the highly processive kinesin. First, there was a critical microtubule length for motility; only microtubules longer than this critical length moved in low density ncd surfaces, which suggested that multiple ncd proteins must cooperate to move microtubules in the surface assay. Under similar conditions, native kinesin demonstrated no critical microtubule length, consistent with the behavior of a highly processive motor. Second, addition of methylcellulose to decrease microtubule diffusion decreased the critical microtubule length for motility. Also, the rates of microtubule motility were microtubule length dependent in methylcellulose; short microtubules, that interacted with fewer ncd proteins, moved more slowly than long microtubules that interacted with more ncd proteins. In contrast, short microtubules, that interacted with one or a few kinesin proteins, moved on average slightly faster than long microtubules that interacted with multiple kinesins. We conclude that a degree of processivity as high as that of kinesin, where a single dimer can move over distances on the order of one micrometer, may not be a general mechanistic feature of the kinesin superfamily. Received: 16 September 1997 / Accepted: 4 November 1997     

小儿细菌性肺炎痰培养物病原菌构成及耐药性分析

目的 分析肺炎患儿痰培养物中细菌构成及其耐药情况,为小儿细菌性肺炎治疗提供合理使用抗感染药物的依据。 方法 对2019年11月至2020年12月济宁市第一人民医院临床送检的肺炎患儿痰液标本进行病原菌的分离鉴定和药敏试验,收集相关资料,并对小儿肺炎病原菌在临床上的分布特点及其耐药情况进一步分析。 结果 痰液培养阳性患儿183例,其中多重细菌感染患儿16例,共培养出199株细菌(剔除同一患儿的重复菌株),其中革兰阴性菌128株(64.32%),革兰阳性菌71株(35.68%)。主流细菌为肺炎链球菌(19.60%)、金黄色葡萄球菌(16.08%)、卡他莫拉菌(13.57%)、肺炎克雷伯菌(11.06%)和大肠埃希菌(10.05%)。多重细菌感染主要以金黄色葡萄球菌+肺炎克雷伯菌、金黄色葡萄球菌+大肠埃希菌混合为主(共5例,31.25%)。肺炎链球菌和金黄色葡萄球菌对利奈唑胺、万古霉素绝对敏感。卡他莫拉菌对头孢噻肟、头孢曲松、米诺环素等绝对敏感。肠杆菌科中肺炎克雷伯菌和大肠埃希菌药敏结果相似,对阿米卡星、厄他培南、头孢替坦敏感率在95%~100%,对氨苄西林耐药率在95%~100%。肺炎链球菌、金黄色葡萄球菌、卡他莫拉菌对左氧氟沙星敏感率均大于90%,大肠埃希菌、肺炎克雷伯菌对左氧氟沙星不够敏感,但中敏率达到75%以上。 结论 肺炎链球菌、金黄色葡萄球菌、卡他莫拉菌、肺炎克雷伯菌、大肠埃希菌感染是本地肺炎患儿痰液中常见菌,抗生素耐药状况比较严重,亚胺培南对革兰阴性菌混合感染的效果较好,利奈唑胺对阳性菌效果较好;左氧氟沙星可用于革兰阴性和阳性菌混合感染,合理使用抗生素应注意针对性和用药时间,杀灭致病菌的同时尽可能维护正常菌群的平衡。     

Interaction of pathogenic fungi with host cells: Molecular and cellular approaches

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host-fungus interaction. Fungi present intra- and/or extracellular host-parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. On the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host-fungus interaction.     

Interaction of pathogenic fungi with host cells: Molecular and cellular approaches

This review provides an overview of several molecular and cellular approaches that are likely to supply insights into the host–fungus interaction. Fungi present intra- and/or extracellular host–parasite interfaces, the parasitism phenomenon being dependent on complementary surface molecules. The entry of the pathogen into the host cell is initiated by the fungus adhering to the cell surface, which generates an uptake signal that may induce its cytoplasmatic internalization. Furthermore, microbial pathogens use a variety of their surface molecules to bind to host extracellular matrix (ECM) components to establish an effective infection. On the other hand, integrins mediate the tight adhesion of cells to the ECM at sites referred to as focal adhesions and also play a role in cell signaling. The phosphorylation process is an important mechanism of cell signaling and regulation; it has been implicated recently in defense strategies against a variety of pathogens that alter host-signaling pathways in order to facilitate their invasion and survival within host cells. The study of signal transduction pathways in virulent fungi is especially important in view of their putative role in the regulation of pathogenicity. This review discusses fungal adherence, changes in cytoskeletal organization and signal transduction in relation to host–fungus interaction.     

Rickettsia Sca2 is a bacterial formin-like mediator of actin-based motility

Diverse intracellular pathogens subvert the host actin-polymerization machinery to drive movement within and between cells during infection. Rickettsia in the spotted fever group (SFG) are Gram-negative, obligate intracellular bacterial pathogens that undergo actin-based motility and assemble distinctive 'comet tails' that consist of long, unbranched actin filaments. Despite this distinct organization, it was proposed that actin in Rickettsia comet tails is nucleated by the host Arp2/3 complex and the bacterial protein RickA, which assemble branched actin networks. However, a second bacterial gene, sca2, was recently implicated in actin-tail formation by R. rickettsii. Here, we demonstrate that Sca2 is a bacterial actin-assembly factor that functionally mimics eukaryotic formin proteins. Sca2 nucleates unbranched actin filaments, processively associates with growing barbed ends, requires profilin for efficient elongation, and inhibits the activity of capping protein, all properties shared with formins. Sca2 localizes to the Rickettsia surface and is sufficient to promote the assembly of actin filaments in cytoplasmic extract. These results suggest that Sca2 mimics formins to determine the unique organization of actin filaments in Rickettsia tails and drive bacterial motility, independently of host nucleators.     

Chromosome architecture is a key element of bacterial cellular organization

The bacterial chromosome encodes information at multiple levels. Beyond direct protein coding, genomes encode regulatory information required to orchestrate the proper timing and levels of gene expression and protein synthesis, and contain binding sites and regulatory sequences to co‐ordinate the activities of proteins involved in chromosome repair and maintenance. In addition, it is becoming increasingly clear that yet another level of information is encoded by the bacterial chromosome – the three‐dimensional packaging of the chromosomal DNA molecule itself and its positioning relative to the cell. This vast structural blueprint of specific positional information is manifested in various ways, directing chromosome compaction, accessibility, attachment to the cell envelope, supercoiling, and general architecture and arrangement of the chromosome relative to the cell body. Recent studies have begun to identify and characterize novel systems that utilize the three‐dimensional spatial information encoded by chromosomal architecture to co‐ordinate and direct fundamental cellular processes within the cytoplasm, providing large‐scale order within the complex clutter of the cytoplasmic compartment.     

PilC of pathogenic Neisseria is associated with the bacterial cell surface

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.     

细菌群集运动特性的研究进展

群集运动(swarming motility)是细菌以群体方式协调性地依靠鞭毛和Ⅳ型菌毛(type Ⅳ pili,TFP)在半固体表面共同运动,是一种典型的协同运动。群集运动因其与生物被膜、子实体的形成、病原体的侵入和微生物的扩散及共生等过程都有着密切的关系而备受人们的关注,是当前微生物领域的一个研究热点。人们对细菌群集运动开展了大量的研究,包括群集运动中关键蛋白表达的变化、细胞间化学交流的变化以及机械性变化等。鞭毛蛋白的表达以及胞内环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)的水平等会对群集运动产生一定的影响,在菌落中复杂地调控着细菌集体行为;群集运动细胞独特的物理性质表现有益于菌落整体的扩张;细菌周围生长环境中的营养和水分含量等因素也在不同程度上影响细菌群集运动的能力。未来,在解析群集运动分子机制的基础上,如何构建一个统一的群集运动模型成为该领域研究面临的一个挑战。     

Spatiotemporal mapping of ex vivo motility in the caecum of the rabbit

We used high definition radial, strain rate and intensity spatiotemporal mapping to quantify contractile movements of the body and associated structures of the rabbit caecum when the terminal ileum was being perfused with saline at a constant rate. This perfusion caused gradual distension of the caecum as a result of relative restriction of outflow from the ampulla caecalis. The body of the caecum exhibited two patterns of motility that appeared autonomous, i.e. occurred independently of any contractile activity at the inlet or outlet. Firstly, the pattern that we termed ladder activity consisted of an orderly sequential contraction of bundles of axially oriented circular muscle between the spiral turns of longitudinal muscle and proceeded either from base to tip or from tip to base at a similar frequency and velocity. Secondly, less-localised, rapidly propagating synchronous contractions of both circular and longitudinal muscle, which were more common when the caecum was distended, that were termed mass peristalsis. Movements of the ileum and sacculus rotundus occurred at the same frequency and were broadly coordinated. Distension of the distal sacculus occurred synchronously with contraction of the ileum and did not propagate in an orderly manner across the structure, i.e. was instantaneous. This pattern was consistent with hydrostatic distension. Contractions propagated through the ampulla caecalis in either an orad or an aborad direction at a similar frequency to, and broadly correlated with, those in the ileum. The frequencies of distension of the sacculus and of contraction in the ileum and ampulla were momentarily augmented during mass peristalsis. The authors conclude that there was some coordination between the contractile activity of the terminal ileum and the caecal ampulla during periods of ongoing inflow from the ileum and between these structures and the caecum during mass peristalsis.     

A conjecture on the relationship of bacterial shape to motility in rod-shaped bacteria

We have calculated the optimal shape, i.e. the length-to-width ratio of a bacterial cell, that allows a bacterial cell to move most efficiently through liquid. For a cell of a given size, a minimum exists in the force required to move through any liquid when the length of the cell is approx. 3.7 times greater than the width. As this is in approximate agreement with the observed shape of bacteria such as the Enterobacteriaceae, we conjecture that the current observed shape of these bacteria may have been determined, in part, to obtain the most efficient shape for moving through liquids. It is also found that spherical cells are very inefficient in movement through liquid, while longer cells of a fixed size are still relatively efficient in moving through liquids. Since the optimal shape is independent of actual size (within large bounds), it is further proposed that hydrodynamic efficiency considerations support the proposal of constant shape over a range of sizes for rod-shaped bacteria.     

Deregulation of transition metals homeostasis is a key feature of cadmium toxicity in Salmonella

Cadmium is a highly toxic metal whose presence in the environment represents a challenge for all forms of life. To improve our knowledge on cadmium toxicity, we have explored Salmonella Typhimurium responses to this metal. We have found that cadmium induces the concomitant expression of the cation efflux pump ZntA and of the high affinity zinc import system ZnuABC. This observation suggests that cadmium accumulation within the cell induces a condition of apparent zinc starvation, possibly due to the ability of this metal to compete with zinc for the metal binding site of proteins. This hypothesis is supported by the finding that strains lacking ZntA or ZnuABC are hyper-susceptible to cadmium and that the cadmium-induced growth defect of a znuABC mutant strain is largely relieved by zinc supplementation. A similar growth defect was observed for a mutant with impaired ability to acquire iron, whereas cadmium does not affect growth of a strain defective in manganese import. Cadmium also influences the expression and activity of the two cytoplasmic superoxide dismutases FeSOD and MnSOD, which are required to control cadmium-mediate oxidative stress. Exposure to cadmium causes a reduction of FeSOD activity in Salmonella wild type and the complete abrogation of its expression in the strain defective in iron import. In contrast, although MnSOD intracellular levels increase in response to cadmium, we observed discrepancies between protein levels and enzymatic activity which are suggestive of incorporation of non-catalytic metals in the active site or to cadmium-mediated inhibition of manganese import. Our results indicate that cadmium interferes with the ability of cells to manage transition metals and highlight the close interconnections between the homeostatic mechanisms regulating the intracellular levels of different metals.