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Bacteria colonize surfaces in the environment and can also penetrate tissues and materials by entering micro- and nano-scale cracks and pores. has been observed within nanoscale channels in bone that are 2-3 times smaller than cell diameter. Inside the bone, bacteria are protected from host immunity and systemic antibiotics, potentially contributing to chronic and recurrent infections. The physical mechanisms that enable bacteria to enter channels smaller than the cell width are unclear. It has been proposed that bacteria traverse narrow passages through division, such that daughter cells form within small channels and proliferate in chains down the channel length. Here, we use microfluidics to test the idea that can traverse submicrometer channels through growth. We examined the net migration of growing cell chains within tapered nanochannels (width ~1.5-0.3 μm). We found that proliferation can facilitate migration, but only to cell deformations of 600 nm (65% cell width). Below 600 nm, mechanical confinement significantly slows or completely inhibits division in single cells. Interestingly, growth arrest occurs independent of the Z-ring assembly and is unrelated to the initial orientation of the division plane. Thus, our findings suggest that it is unlikely for to traverse nanoscale channels via division. Bacteria that colonize materials and tissues within the body can be difficult to remove, even with thorough cleaning and application of antibiotics. Recent studies show that bacteria not only colonize the surfaces of tissues in the body but can also squeeze into naturally occurring pores and channels and thereby gain protection from immune cells and antibiotics. Here, we ask how physical forces and cell growth might enable bacteria to enter small pores within materials. We use microfluidic devices to study the growth and migration of the human pathogenic bacteria, .

Inflammatory bowel disease (IBD) is a multifactorial disease which arises as a result of the interaction of genetic, environmental, barrier and microbial factors leading to chronic inflammation in the intestine. Patients with IBD had a higher risk of developing colorectal carcinoma (CRC), of which the subset was classified as colitis-associated cancers. Genetic polymorphism of innate immune receptors had long been considered a major risk factor for IBD, and the mutations were also recently observed in CRC. Altered microbial composition (termed microbiota dybiosis) and dysfunctional gut barrier manifested by epithelial hyperpermeability and high amount of mucosa-associated bacteria were observed in IBD and CRC patients. The findings suggested that aberrant immune responses to penetrating commensal microbes may play key roles in fueling disease progression. Accumulative evidence demonstrated that mucosa-associated bacteria harbored colitogenic and protumoral properties in experimental models, supporting an active role of bacteria as pathobionts (commensal-derived opportunistic pathogens). Nevertheless, the host factors involved in bacterial dysbiosis and conversion mechanisms from lumen-dwelling commensals to mucosal pathobionts remain unclear. Based on the observation of gut leakiness in patients and the evidence of epithelial hyperpermeability prior to the onset of mucosal histopathology in colitic animals, it was postulated that the epithelial barrier dysfunction associated with mucosal enrichment of specific bacterial strains may predispose the shift to disease-associated microbiota. The speculation of leaky gut as an initiating factor for microbiota dysbiosis that eventually led to pathological consequences was proposed as the “common ground hypothesis”, which will be highlighted in this review. Overall, the understanding of the core interplay between gut microbiota and epithelial barriers at early subclinical phases will shed light to novel therapeutic strategies to manage chronic inflammatory disorders and colitis-associated cancers.

Staphylococcus aureus is a pathogen associated with severe respiratory infections. The ability of S. aureus to internalize into lung epithelial cells complicates the treatment of respiratory infections caused by this bacterium. In the intracellular environment, S. aureus can avoid elimination by the immune system and the action of circulating antibiotics. Consequently, interfering with S. aureus internalization may represent a promising adjunctive therapeutic strategy to enhance the efficacy of conventional treatments. Here, we investigated the host-pathogen molecular interactions involved in S. aureus internalization into human lung epithelial cells. Lipid raft-mediated endocytosis was identified as the main entry mechanism. Thus, bacterial internalization was significantly reduced after the disruption of lipid rafts with methyl-β-cyclodextrin. Confocal microscopy confirmed the colocalization of S. aureus with lipid raft markers such as ganglioside GM1 and caveolin-1. Adhesion of S. aureus to α5β1 integrin on lung epithelial cells via fibronectin-binding proteins (FnBPs) was a prerequisite for bacterial internalization. A mutant S. aureus strain deficient in the expression of alpha-hemolysin (Hla) was significantly impaired in its capacity to enter lung epithelial cells despite retaining its capacity to adhere. This suggests a direct involvement of Hla in the bacterial internalization process. Among the receptors for Hla located in lipid rafts, caveolin-1 was essential for S. aureus internalization, whereas ADAM10 was dispensable for this process. In conclusion, this study supports a significant role of lipid rafts in S. aureus internalization into human lung epithelial cells and highlights the interaction between bacterial Hla and host caveolin-1 as crucial for the internalization process.

is a facultative intracellular Gram-negative bacterium, responsible for a wide range of food- and water-borne diseases ranging from gastroenteritis to typhoid fever depending on hosts and serotypes. thus represents a major threat to public health. A key step in pathogenesis is the invasion of phagocytic and non-phagocytic host cells. To trigger its own internalization into non-phagocytic cells, has developed different mechanisms, involving several invasion factors. For decades, it was accepted that could only enter cells through a type three secretion system, called T3SS-1. Recent research has shown that this bacterium expresses outer membrane proteins, such as the Rck protein, which is able to induce entry mechanism. Rck mimics natural host cell ligands and triggers engulfment of the bacterium by interacting with the epidermal growth factor receptor. is thus able to use multiple entry pathways during the infection process. However, it is unclear how and when exploits the T3SS-1 and Rck entry mechanisms. As a series of reviews have focused on the T3SS-1, this review aims to describe the current knowledge and the limitations of our understanding of the Rck outer membrane protein. The regulatory cascade which controls Rck expression and the molecular mechanisms underlying Rck-mediated invasion into cells are summarized. The potential role of Rck-mediated invasion in pathogenesis and the intracellular behavior of the bacteria following a Rck-dependent entry are discussed.

Background During autophagy defense against invading microbes, certain lipid types are indispensable for generating specialized membrane-bound organelles. The lipid composition of autophagosomes remains obscure, as does the issue of how specific lipids and lipid-associated enzymes participate in autophagosome formation and maturation. Helicobacter pylori is auxotrophic for cholesterol and converts cholesterol to cholesteryl glucoside derivatives, including cholesteryl 6ʹ- O -acyl-α -d- glucoside (CAG). We investigated how CAG and its biosynthetic acyltransferase assist H. pylori to escape host-cell autophagy. Methods We applied a metabolite-tagging method to obtain fluorophore-containing cholesteryl glucosides that were utilized to understand their intracellular locations. H. pylori 26695 and a cholesteryl glucosyltransferase (CGT)-deletion mutant (ΔCGT) were used as the standard strain and the negative control that contains no cholesterol-derived metabolites, respectively. Bacterial internalization and several autophagy-related assays were conducted to unravel the possible mechanism that H. pylori develops to hijack the host-cell autophagy response. Subcellular fractions of H. pylori- infected AGS cells were obtained and measured for the acyltransferase activity. Results The imaging studies of fluorophore-labeled cholesteryl glucosides pinpointed their intracellular localization in AGS cells. The result indicated that CAG enhances the internalization of H. pylori in AGS cells. Particularly, CAG, instead of CG and CPG, is able to augment the autophagy response induced by H. pylori. How CAG participates in the autophagy process is multifaceted. CAG was found to intervene in the degradation of autophagosomes and reduce lysosomal biogenesis, supporting the idea that intracellular H. pylori is harbored by autophago-lysosomes in favor of the bacterial survival. Furthermore, we performed the enzyme activity assay of subcellular fractions of H. pylori -infected AGS cells. The analysis showed that the acyltransferase is mainly distributed in autophago-lysosomal compartments. Conclusions Our results support the idea that the acyltransferase is mainly distributed in the subcellular compartment consisting of autophagosomes, late endosomes, and lysosomes, in which the acidic environment is beneficial for the maximal acyltransferase activity. The resulting elevated level of CAG can facilitate bacterial internalization, interfere with the autophagy flux, and causes reduced lysosomal biogenesis.

Successful intracellular replication is a defining feature of many bacterial pathogens and directly influences disease outcome. For the salmonid pathogen Piscirickettsia salmonis , the intracellular environment that supports bacterial growth has remained incompletely characterized. Here, we show that P. salmonis replicates within an acidified, Lamp-1–positive vacuole and that intracellular growth is influenced by host iron availability. Infection is accompanied by activation of lysosomal pathways in host cells and coordinated induction of bacterial stress-response mechanisms, secretion systems, iron-acquisition pathways, and numerous genes of previously unknown function. Intracellular passage also alters bacterial behavior during subsequent infection cycles, suggesting a physiological adaptation associated with host-cell residence. By defining the intracellular context in which P. salmonis proliferates and situating these features within the broader landscape of intracellular bacterial strategies, this work advances understanding of host–pathogen interactions in non-mammalian systems and provides a foundation for future functional studies relevant to aquaculture and intracellular microbiology.

Diverse microbes engage CD13 to infect host cells. Yet invasin-CD13 interactions, the signaling they invoke for pathogen entry, and the relevance of CD13 to infection in vivo are underexplored. Dissecting these concepts would advance fundamental understanding of a convergently evolved infection strategy and could have translational benefits. Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging disease for which there is no vaccine and few therapeutic options. We found that A. phagocytophilum uses its surface protein and recently identified protective immunogen, AipA, to bind CD13 to elicit Src kinase signaling, which is critical for infection. We elucidated the AipA CD13 binding domain, which CD13 region AipA engages, and established that CD13 is key for A. phagocytophilum infection in vivo. Disrupting the AipA-CD13 interaction could be utilized to prevent granulocytic anaplasmosis and offers a model that could be applied to protect against multiple infectious diseases.

Chlamydia trachomatis is a human pathogen and a prevalent agent of sexually transmitted diseases. The ability to survive and propagate within a protected intracellular niche leads directly to pathology indicative of Chlamydia -mediated disease. The reduced chlamydial genome leads to comparatively limited biosynthetic capacity, thereby necessitating parasitism of metabolites and other resources from the infected host cell. Chlamydia relies heavily on type III secreted effectors to interface with and co-opt host pathways to acquire resources. We demonstrate herein that the plasma membranes of infected cells represent a potential reservoir of resources required for optimal intracellular growth. Chlamydiae employ at least one type III secreted effector protein, translocated membrane-associated effector A (TmeA), to redirect material to the vacuole by manipulating Arp2/3-dependent actin polymerization. This pathway represents a distinct mechanism by which Chlamydia acquires resources and provides evidence for TmeA function during intracellular development.

Complete eradication of bacterial infections is often a challenging task, especially in presence of prosthetic devices. Invasion of non-phagocytic host cells appears to be a critical mechanism of microbial persistence in host tissues. Hidden within host cells, bacteria elude host defences and antibiotic treatments that are intracellularly inactive. The intracellular invasiveness of bacteria is generally measured by conventional gentamicin protection assays. The efficiency of invasion, however, markedly differs across bacterial species and adjustments to the titre of the microbial inocula used in the assays are often needed to enumerate intracellular bacteria. Such changes affect the standardisation of the method and hamper a direct comparison of bacteria on a same scale. This study aims at investigating the precise relation between inoculum, in terms of multiplicity of infection (MOI), and internalised bacteria. The investigation included nine Staphylococcus aureus, seven Staphylococcus epidermidis, five Staphylococcus lugdunensis and two Enterococcus faecalis clinical strains, which are co-cultured with MG63 human osteoblasts. Unprecedented insights are offered on the relations existing between MOI, number of internalised bacteria and per cent of internalised bacteria. New parameters are identified that are of potential use for qualifying the efficiency of internalization and compare the behaviour of bacterial strains.

Once inside, the immigrant anaerobic or facultative bacteria can find refuge in nutrient-rich, hypoxic regions of the tumor tissues while remaining dormant for years, evading immune clearance by hiding in the body’s own cancer cells, and can become pathogenic as they find an immunologically favorable environment. A recent article in the journal Science demonstrated that the presence of bacteria inside tumor have also the potential to create resistance against cancer therapy due to their inherent capability of cleaving chemotherapeutics into inactive metabolites (5). A 2016 study by Din et al. showed how a clinically relevant bacterium could be engineered using tools of synthetic biology to lyse synchronously at a threshold population density and release genetically encoded anticancer cargo on demand in the required vicinity (26). Singh R, Patil S, Singh N, Gupta S. Dual functionality nanobioconjugates targeting intracellular bacteria in cancer cells with enhanced antimicrobial activity.