How Bacteria Subvert Animal Cell Structure and Function.

Bacterial pathogens encode a wide variety of effectors and toxins that hijack host cell structure and function. Of particular importance are virulence factors that target actin cytoskeleton dynamics critical for cell shape, stability, motility, phagocytosis, and division. In addition, many bacteria target organelles of the general secretory pathway (e.g., the endoplasmic reticulum and the Golgi complex) and recycling pathways (e.g., the endolysosomal system) to establish and maintain an intracellular replicative niche. Recent research on the biochemistry and structural biology of bacterial effector proteins and toxins has begun to shed light on the molecular underpinnings of these host-pathogen interactions. This exciting work is revealing how pathogens gain control of the complex and dynamic host cellular environments, which impacts our understanding of microbial infectious disease, immunology, and human cell biology.

Bacterial pathogen manipulation of host membrane trafficking.

Pathogens use a vast number of strategies to alter host membrane dynamics. Targeting the host membrane machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Membrane manipulation promotes bacterial replication while suppressing host responses, allowing the bacterium to thrive in a hostile environment. This review provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack host membrane trafficking machinery. We start with mechanisms used by bacteria to alter the plasma membrane, delve into the hijacking of various vesicle trafficking pathways, and conclude by summarizing bacterial adaptation to host immune responses. Understanding bacterial manipulation of host membrane trafficking provides insights into bacterial pathogenesis and uncovers the molecular mechanisms behind various processes within a eukaryotic cell.

Overview and recent developments in cell-based noninvasive prenatal testing.

Investigators have long been interested in the natural phenomenon of fetal and placental cell trafficking into the maternal circulation. The scarcity of these circulating cells makes their detection and isolation technically challenging. However, as a DNA source of fetal origin not mixed with maternal DNA, they have the potential of considerable benefit over circulating cell-free DNA-based noninvasive prenatal genetic testing (NIPT). Endocervical trophoblasts, which are less rare but more challenging to recover are also being investigated as an approach for cell-based NIPT. We review published studies from around the world describing both forms of cell-based NIPT and highlight the different approaches' advantages and drawbacks. We also offer guidance for developing a sound cell-based NIPT protocol.

Imaging aquatic animal cells and associated pathogens by atomic force microscopy in air.

Atomic force microscopy (AFM) is a sophisticated imaging tool with nanoscale resolution that is widely used in structural biology, cell biology, and material science, among other fields. However, to date it has rarely been applied to the study of aquatic animals, especially on one of the main cultured species, shrimp. One reason for this is that no shrimp cell line established until now, primary cell is fragile and difficult to be studied under AFM. In this study, we used AFM to image three different types of biological material from shrimp (Litopenaeus vannamei) in air, including hemocytes and two associated pathogens. Without obvious deformations when the cells were imaged in air and in the case for the haemocytes and the cells were fixed as well. The result suggests hydrophobic glass coverslips are a suitable substrate for adhesion of these samples. The method described here can be applied to the preparation of other fragile biological samples from aquatic animals for high-resolution analyses of host-pathogen interactions and other basic physiological processes.

Autophagosome formation in response to intracellular bacterial invasion.

Autophagy is an intracellular bulk degradation system in which double-membrane vesicles, called autophagosomes, engulf cytoplasmic components and later fuse with lysosomes to degrade the autophagosome content. Although autophagy was initially thought a non-selective process, recent studies have clarified that it can selectively target intracellular bacteria and function as an intracellular innate immune system that suppresses bacterial survival. A key mechanism for the recognition of cytosol-invading bacteria is ubiquitination, and the recognition of the ubiquitinated target by the autophagy machinery can be accomplished multiple ways. In this review, we discuss recent findings regarding the induction of autophagosome formation in response to intracellular bacterial invasion.

Interaction of invasive bacteria with host signaling pathways.

Many bacterial pathogens exploit mammalian cell functions in order to promote their adherence to or uptake by host cells. Recent work has led to the identification of some of the bacterial and mammalian proteins involved in these processes. Although specific mechanisms differ among pathogens, a common aspect appears to be regulation of signaling pathways that control the actin cytoskeleton.

[Autophagy and pathogens: «Bon appétit Messieurs!¼]. / Autophagie et pathogènes: «Bon appétit Messieurs!¼

Autophagy is a highly conserved, self-degradative pathway for clearance and recycling of cytoplasmic contents. This ubiquitous cell intrinsic process can be used as a defence mechanism against intracellular pathogens. Indeed autophagy is increased upon pathogen detection, and experimental extinction in vitro and in vivo of this cellular process has been demonstrated as a crucial role to control intracellular pathogens. Co-evolution between host-cells and pathogens has selected numerous micoorganisms able to avoid or usurp autophagy to their own benefit. Understanding mechanisms underlying the anti-microbial properties of autophagy as well as those used by certain pathogens to escape this cellular process might be crucial to manipulate this cellular function in order to prevent or treat infectious diseases.

Enzyme Activity of Cellulolytic Bacteria from Biological Education and Research Forest Floor Andalas University.

<b>Background and Objective:</b> The composition of the waste consists mostly of plant biomass. Cellulose is the largest component of plant biomass and cellulolytic bacteria are needed to degrade it. This study aimed to determine enzyme activity possessed by bacterial isolates from Biological Education and Research Forest floor Andalas University. <b>Materials and Methods:</b> The isolation stage was carried out with NA (Nutrient agar) medium, Screening with CMC (Carboxymethyl Cellulose) medium with congo red dye and enzyme activity testing was carried out using the Nelson-Somogyi method. <b>Results:</b> We found 16 bacterial isolates obtained from Biological Education and Research Forest Floor Andalas University, 10 of them were positive for cellulolytic bacteria with the highest cellulolytic index value of 2.59 on FFB 2 isolates. <b>Conclusion:</b> The bacterial isolate with the best enzyme activity value was FFB 2 isolate 0.166 U mL¹ for 72 hrs.

Detection of Mollicutes in bioreactor samples by real-time transcription-mediated amplification.

AIM: Contamination by Mollicutes is a significant challenge for research laboratories and biopharmaceutical industry. It leads to alteration of results or production quality as well as loss of time, materials and revenue. These organisms can czoriginate from mammalian, avian, insect, plant or fish cells. Culture-based methods may require 28 days to detect Mollicutes. Traditional microbiology could advantageously be replaced by nucleic acid testing for earlier detection. METHODS AND RESULTS: A membrane filtration-based concentration of the Mollicutes has been coupled to real-time transcription-mediated amplification (real-time TMA) to demonstrate these advantages. The eight species required by European Pharmacopoeia have been tested and were detected with sensitivity below 100 CFU per 20-ml sample. Co-culture experiments, in which Mollicutes are grown with CHO-S (suspension) or HEK 293 (adherent) cells, were also performed to respectively mimic a bioreactor or flask contamination. Despite the fact that Mollicutes can attach to or invade mammalian cells, they were consistently detected over multiple days. CONCLUSIONS: the sample preparation and amplification method used in this study increases sensitivity and reduces time-to-result for detection of Mollicutes. SIGNIFICANCE AND IMPACT OF THE STUDY: the described system allows real-time monitoring for microbial contamination of cell-based processes and products for the biopharmaceutical industry.

Insects as hosts for mutualistic bacteria.

Insects are among the most successful animals on Earth both with regard to their biomass and biodiversity. It is estimated that up to 20% of all insects are obligately associated with symbiotic microorganisms, and it is likely that their capacity to engage microbial companions has greatly contributed to their evolutionary success. The main focus of this review lies on obligately intracellular bacteria residing in specialized cells, the bacteriocytes, provided by the host. In the past few years the focus in research on these bacteria has been on their biological role for the host and the consequences on the genome and metabolic capacities shaped by a long-lasting obligate association confined to the interior of a eukaryotic host cell. Here, we compare those endosymbiont-host interactions where the genome of the bacterium is sequenced.

How cells clean house.

Inside the cytoplasm of a living cell, organelles called autophagosomes continually engulf bits of cytoplasm, along with damaged cell parts and invading bacteria and viruses. The "sweepings" are carried to digestive organelles for breakup and recycling. The process is called autophagy. Cell biologists are learning about autophagy in great detail by tracing the protein signals that drive and control the process. A fuller understanding of autophagy is opening up new options for treating cancer, infectious disease, immune disorders and dementia, and it may one day even help to slow down aging.

Ortholog-based screening and identification of genes related to intracellular survival.

Bioinformatics and comparative genomics analysis methods were used to predict unknown pathogen genes based on homology with identified or functionally clustered genes. In this study, the genes of common pathogens were analyzed to screen and identify genes associated with intracellular survival through sequence similarity, phylogenetic tree analysis and the λ-Red recombination system test method. The total 38,952 protein-coding genes of common pathogens were divided into 19,775 clusters. As demonstrated through a COG analysis, information storage and processing genes might play an important role intracellular survival. Only 19 clusters were present in facultative intracellular pathogens, and not all were present in extracellular pathogens. Construction of a phylogenetic tree selected 18 of these 19 clusters. Comparisons with the DEG database and previous research revealed that seven other clusters are considered essential gene clusters and that seven other clusters are associated with intracellular survival. Moreover, this study confirmed that clusters screened by orthologs with similar function could be replaced with an approved uvrY gene and its orthologs, and the results revealed that the usg gene is associated with intracellular survival. The study improves the current understanding of intracellular pathogens characteristics and allows further exploration of the intracellular survival-related gene modules in these pathogens.

Subtle changes in host cell density cause a serious error in monitoring of the intracellular growth of Chlamydia trachomatis in a low-oxygen environment: Proposal for a standardized culture method.

We monitored Chlamydia trachomatis growth in HeLa cells cultured with either DMEM or RPMI medium containing 10% FCS under 2% or 21% O2 conditions for 2 days. Bacterial numbers, host cell numbers, and fibrosis-related gene expression in the host cells were estimated by an inclusion forming unit assay, a cell counting assay, and a PCR array, respectively. In contrast to RPMI, bacterial growth under low oxygen conditions in DMEM rapidly decreased with increasing host cell density. The addition of supplements (glucose, glutamine, vitamin B12, D-biotin, non-essential amino acids, glutathione) to the media had no effect. The growth of host cells in DMEM under low oxygen conditions rapidly decreased, although the cells remained healthy morphologically. Furthermore, the downregulation of 17 genes was observed under low oxygen in DMEM. Whereas no effect on bacterial growth was observed when culturing in RPMI medium at low oxygen, and the downregulation of three genes (CTGF, SERPINE1, JUN) was observed following bacterial infection compared with the uninfected control cells. Thus, our findings indicate the need for carefully selected culture conditions when performing experiments with C. trachomatis under low-oxygen environments, and RPMI (rather than DMEM) is recommended when a low host cell density is to be used, proposing the major modification of cell culturing method of C. trachomatis in a low-oxygen environment.

Salmonella Populations inside Host Cells.

Bacteria of the Salmonella genus cause diseases ranging from gastroenteritis to life-threatening typhoid fever and are among the most successful intracellular pathogens known. After the invasion of the eukaryotic cell, Salmonella exhibits contrasting lifestyles with different replication rates and subcellular locations. Although Salmonella hyper-replicates in the cytosol of certain host cell types, most invading bacteria remain within vacuoles in which the pathogen proliferates at moderate rates or persists in a dormant-like state. Remarkably, these cytosolic and intra-vacuolar intracellular lifestyles are not mutually exclusive and can co-exist in the same infected host cell. The mechanisms that direct the invading bacterium to follow the cytosolic or intra-vacuolar "pathway" remain poorly understood. In vitro studies show predominance of either the cytosolic or the intra-vacuolar population depending on the host cell type invaded by the pathogen. The host and pathogen factors controlling phagosomal membrane integrity and, as consequence, the egress into the cytosol, are intensively investigated. Other aspects of major interest are the host defenses that may affect differentially the cytosolic and intra-vacuolar populations and the strategies used by the pathogen to circumvent these attacks. Here, we summarize current knowledge about these Salmonella intracellular subpopulations and discuss how they emerge during the interaction of this pathogen with the eukaryotic cell.

The intracellular life of Chlamydia psittaci: how do the bacteria interact with the host cell?

Throughout the life of any organism interactions with the surrounding environment are always taking place, a process that leads to evolution. Chlamydia psittaci is an obligate intracellular parasite, but it must also be capable of extracellular survival in order to search for new host cells. Therefore, these peculiar prokaryotes have evolved two different particles and a unique developmental cycle that, together with a series of not yet fully understood interactions with their host cells, allow them to fulfil the requirements for their permanence in nature. These interactions are the subject of this paper. Particular attention is paid to the attachment and internalization of the bacteria, the chlamydial vacuole, and the avoidance of lysosomal degradation.

Determinants of cell entry and intracellular survival of Mycobacterium tuberculosis.

The ability of Mycobacterium tuberculosis to sustain a chronic infection and cause disease in a subset of those infected depends on its products--virulence factors--that enable the organism to enter and survive indefinitely inside mononuclear phagocytic cells by subverting cellular antimicrobial mechanisms. Characterizing these factors is essential to understanding the pathogenesis of M. tuberculosis.

Antibody-mediated protection against intracellular pathogens.

The view that antibody-mediated protection is unimportant against intracellular pathogens is not supported by the literature. In fact, there is convincing evidence that antibody can protect against many important intracellular pathogens. The challenge now is to identify antigens that elicit protective antibodies, use them in vaccine design and understand how humoral and cellular immune mechanisms cooperate.

Interaction of Yersinia enterocolitica biotype 1A strains of diverse origin with cultured cells in vitro.

Yersinia enterocolitica biotype 1A isolates are increasingly being associated with diarrhea. However, the mechanism of their pathogenicity is not well understood. In the present study interaction of Y. enterocolitica isolates with CHO cells, HEp-2 cells and J774 mouse macrophages was studied. Y. enterocolitica biotype 1A strains of clinical origin invaded CHO and HEp-2 cells to a significantly higher degree than non-clinical isolates. However, among non-clinical isolates, Y. enterocolitica strains of swine origin showed significantly more invasion in CHO and HEp-2 cells than water isolates. Y. enterocolitica isolates from clinical samples exhibited a greater level of survival in macrophages than isolates from non-clinical sources. It may be construed that Y. enterocolitica biotype 1A isolates of clinical and swine origin have higher virulence potential than those from other sources.

Caseinoglycomacropeptide inhibits adhesion of pathogenic Escherichia coli strains to human cells in culture.

Caseinoglycomacropeptide (CGMP) derived from kappa-casein was investigated for its ability to inhibit the adhesion of 3 strains of verotoxigenic Escherichia coli (VTEC) and 3 strains of enteropathogenic Escherichia coli (EPEC) to human HT29 tissue cell cultures. Effects on adhesion of Desulfovibrio desulfuricans, Lactobacillus pentosus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus gasseri were also investigated. Generally, CGMP exerted effective anti-adhesive properties at a dose of 2.5 mg/mL, albeit with a high degree of strain specificity. The CGMP reduced adhesion of VTEC strains to <50% of the control and reduced adhesion of EPEC strains to between 80 and 10% of the control. The CGMP also reduced the adhesion of L. pentosus and L. casei to 44 and 42%, respectively. A slight but significant reduction of L. acidophilus, to 81%, was observed, but no significant effects were detected with either Dsv. desulfuricans or L. gasseri. Further investigation of the dose response relationships with the E. coli strains gave IC50 values ranging between 0.12 and 1.06 mg/mL.