Intracellular fate of strains of Escherichia coli isolated from dairy cows with acute or chronic mastitis.

Research on mastitis in dairy cows caused by Escherichia coli has reported the emergence of strains capable of inducing chronic mastitis and that these strains adhered to and internalized into bovine mammary epithelial cells better than strains of E. coli isolated from acute mastitis. To understand mechanisms and strategies used by chronic E. coli strains to survive intracellularly internalization studies using bovine mammary epithelial cells treated with inhibitors of caveolae-mediated endocytosis (CME) and receptor-mediated endocytosis (RME), double immunofluorescence labeling confocal laser and fluorescence microscopy were conducted. Internalization studies showed that strains chronic E. coli strains persisted intracellularly longer than acute E. coli strains. Treatment of bovine mammary epithelial cells CME or RME inhibitors resulted in lower numbers of intracellular E. coli strains associated with chronic or acute mastitis than untreated controls. In addition, when selective CME inhibitors were used significantly fewer chronic E. coli were detected intracellularly than acute E. coli or untreated controls. Confocal laser microscopy showed that chronic E. coli strains colocalized preferentially with caveolae whereas acute strains did so with early endosomes, an early step of RME. These results suggest that strains of E. coli associated with chronic mastitis exploit lipid rafts/CME to internalize into and move through mammary epithelial cells. By exploiting this endocytosis pathway, chronic E. coli strains avoid bactericidal mechanisms such as endosome acidification and endosome-lysosome fusion, thus allowing intracellular survival. Data from this study helps to explain how these strains are capable of causing chronic E. coli mastitis.

Caveolae-mediated entry of Salmonella typhimurium in a human M-cell model.

Intestinal M cells in Peyer's patches, the specialized antigen-sampling cells of the mucosal immune system, are exploited by Salmonella and other pathogens as a route of invasion. Thus, M cells have attracted lots of attention as a major target of the mucosal immune system. Here, we report that caveolin-1 plays a crucial role in the entry of Salmonella into M cells. We established an in vitro M-like cell model in which polarized enterocyte-like Caco-2 cells created after co-culturing with the Raji B cell line that underwent a phenotypic switch to a form that morphologically and functionally resembles the specialized antigen-transporting M cells. Caveolin-1 was highly expressed in the M-like cells, while not in Caco-2 cells, and a great number of Salmonella infected caveolin-1-expressing M-like cells. To elucidate the role of caveolin-1 in the entry of Salmonella, we downregulated caveolin-1 expression by siRNA and analyzed the level of Salmonella transcytosis across the M-like cells. Transcytosis of Salmonella was markedly reduced by downregulation of caveolin-1 in the M-like cells. These results suggest that caveolin-1 is implicated in the gateway of microbial pathogens through M cells, and, thus, provides a new target of mucosal immunity.

Campylobacter jejuni survives within epithelial cells by avoiding delivery to lysosomes.

Campylobacter jejuni is one of the major causes of infectious diarrhea world-wide, although relatively little is know about its mechanisms of pathogenicity. This bacterium can gain entry into intestinal epithelial cells, which is thought to be important for its ability to persistently infect and cause disease. We found that C. jejuni is able to survive within intestinal epithelial cells. However, recovery of intracellular bacteria required pre-culturing under oxygen-limiting conditions, suggesting that C. jejuni undergoes significant physiological changes within the intracellular environment. We also found that in epithelial cells the C. jejuni-containing vacuole deviates from the canonical endocytic pathway immediately after a unique caveolae-dependent entry pathway, thus avoiding delivery into lysosomes. In contrast, in macrophages, C. jejuni is delivered to lysosomes and consequently is rapidly killed. Taken together, these studies indicate that C. jejuni has evolved specific adaptations to survive within host cells.

Invasion mechanisms of Gram-positive pathogenic cocci.

Gram-positive cocci are important human pathogens. Streptococci and staphylococci in particular are a major threat to human health, since they cause a variety of serious invasive infections. Their invasion into normally sterile sites of the host depends on elaborated bacterial mechanisms that involve adhesion to the host tissue, its degradation, internalisation by host cells, and passage through epithelia and endothelia. Interactions of bacterial surface proteins with proteins of the host's extracellular matrix as well as with cell surface receptors are crucial factors in these processes, and some of the key mechanisms are similar in many pathogenic Gram-positive cocci. Therapies that interfere with these mechanisms may become efficient alternatives to today's antibiotic treatments.

"Alternative" endocytic mechanisms exploited by pathogens: new avenues for therapeutic delivery?

Some pathogens utilize unique routes to enter cells that may evade the intracellular barriers encountered by the typical clathrin-mediated endocytic pathway. Retrograde transport and caveolar uptake are among the better characterized pathways, as alternatives to clathrin-mediated endocytosis, that are known to facilitate entry of pathogens and potential delivery agents. Recent characterization of the trafficking mechanisms of prion proteins and certain bacteria may present new paradigms for strategizing improvements in therapeutic spread and retention of therapy. This review will provide an overview of such endocytic pathways, and discuss current and future possibilities in using these routes as a means to improve therapeutic delivery.

Trafficking of Streptococcus uberis in bovine mammary epithelial cells.

Results from our laboratory showed that Streptococcus uberis internalized bovine mammary epithelial cells by exploiting host cell cytoskeleton and signal transduction mechanisms. It was also shown that S. uberis survived intracellularly for up to 120 h and capable of transcytose bovine mammary epithelial cells. To define mechanisms and strategies used by S. uberis to move through host cells and survive intracellularly, internalization studies using specific inhibitors, double immunofluorescence labeling and confocal laser microscopy were conducted. When bovine mammary epithelial cells were treated with inhibitors of endocytic vesicle acidification, the number of intracellular S. uberis was similar to untreated controls. When selective inhibitors of lipid rafts/caveolae or receptor-mediated endocytosis were used, a significantly lower number of intracellular S. uberis was detected compared with untreated controls. However, when the effect of inhibitors of receptor-mediated endocytosis and lipid rafts/caveolae were compared, the latter induced the lowest S. uberis internalization values suggesting a preferential exploitation of caveolae-mediated endocytosis. Since caveloae-dependent intracellular trafficking does not include intravesicular acidification or lysosome fusion; these results suggest that by exploiting preferential intracellular trafficking pathways in bovine mammary epithelial cells, S. uberis avoids intracellular bactericidal mechanisms. Such a strategy would allow S. uberis to persist intracellularly and may explain how persistent intramammary infections occur.

Signal transduction events involved in human epithelial cell invasion by Campylobacter jejuni 81-176.

Analyses of invasive enteric bacteria (e.g. Shigella, Salmonella, Listeria, and Campylobacter) have shown that these pathogens initiate orchestrated signal transduction cascades in host cells leading to host cytoskeletal rearrangements that result in bacterial uptake. This current study was specifically aimed at examining the involvement of host membrane caveolae and certain protein kinases in epithelial cell invasion by C. jejuni strain 81-176, for which we have previously characterized the kinetics of entry and a unique microtubule-dependent mechanism of internalization. Utilizing in vitro cultured cell invasion assays with a gentamicin-kill step, disruption of membrane caveolae by pretreatment of INT407 cell monolayers with filipin III reduced C. jejuni 81-176 entry by >95%. Strain 81-176 uptake into INT407 cells was markedly inhibited by monolayer pretreatment with the protein kinase inhibitors genistein and staurosporine, or specific inhibitors of PI 3-kinase, wortmannin and LY294002. Western blot analysis using monoclonal anti-protein tyrosine phosphorylation antibody revealed distinctive changes during invasion in phosphorylation of at least nine proteins. Further inhibitor studies indicated that heterotrimeric G proteins, plus ERK and p38 MAP kinase activation are also involved in C. jejuni 81-176 invasion. These results suggest that C. jejuni 81-176 interact at host cell surface membrane caveolae with G protein-coupled receptors, which presumably trigger G-proteins and kinases to activate host proteins including PI 3-kinase and MAP kinases, that appear to be intimately involved in the events controlling 81-176 internalization.

The role of lipid rafts in the pathogenesis of bacterial infections.

Numerous pathogens have evolved mechanisms of co-opting normal host endocytic machinery as a means of invading host cells. While numerous pathogens have been known to enter cells via traditional clathrin-coated pit endocytosis, a growing number of viral and bacterial pathogens have been recognized to invade host cells via clustered lipid rafts. This review focuses on several bacterial pathogens that have evolved several different mechanisms of co-opting clustered lipid rafts to invade host cells. Although these bacteria have diverse clinical presentations and many differences in their pathogenesis, they each depend on the integrity of clustered lipid rafts for their intracellular survival. Bacterial invasion via clustered lipid rafts has been recognized as an important virulence factor for a growing number of bacterial pathogens in their battle against host defenses.

Lipid rafts, caveolae, caveolin-1, and entry by Chlamydiae into host cells.

Obligate intracellular bacterial pathogens of the genus Chlamydia are reported to enter host cells by both clathrin-dependent and clathrin-independent processes. C. trachomatis serovar K recently was shown to enter cells via caveolae-like lipid raft domains. We asked here how widespread raft-mediated entry might be among the Chlamydia. We show that C. pneumoniae, an important cause of respiratory infections in humans that additionally is associated with cardiovascular disease, and C. psittaci, an important pathogen in domestic mammals and birds that also infects humans, each enter host cells via cholesterol-rich lipid raft microdomains. Further, we show that C. trachomatis serovars E and F also use these domains to enter host cells. The involvement of these membrane domains in the entry of these organisms was indicated by the sensitivity of their entry to the raft-disrupting agents Nystatin and filipin, and by their intracellular association with caveolin-1, a 22-kDa protein associated with the formation of caveolae in rafts. In contrast, caveolin-marked lipid raft domains do not mediate entry of C. trachomatis serovars A, 36B, and C, nor of LGV serovar L2 and MoPn. Finally, we show that entry of each of these chlamydial strains is independent of cellular expression of caveolin-1. Thus, entry via the Nystatin and filipin-sensitive pathway is dependent on lipid rafts containing cholesterol, rather than invaginated caveolae per se.

Host cell caveolae act as an entry-port for group A streptococci.

This study identified caveolae as an entry port for group A streptococci into epithelial and endothelial cells. Scanning electron microscopy as well as ultrathin sections of infected cells demonstrated accumulation of small omega-shaped cavities in the host cell membrane close to adherent streptococci. During invasion, invaginations were formed that subsequently revealed intracellular compartments surrounding streptococci. Caveolin-1 was shown to be present in the membrane of invaginations and the compartment membranes. These compartments were devoid of any classic endosomal/lysosomal marker proteins and can thus be described as caveosomes. Disruption of caveolae with methyl-beta-cyclodextrin and filipin abolished host cell invasion. Importantly, streptococci inside caveosomes avoid fusion with lysosomes. Expressing of SfbI protein on the surface of the non-invasive S. gordonii resulted in identical morphological alterations on the host cell as for S. pyogenes. Incubation of HUVEC cells with purified recombinant sole SfbI protein also triggered accumulation of cavity-like structures and formation of membrane invaginations. Tagged to colloidal gold-particles, SfbI protein was shown to cluster following membrane contact. Thus, our results demonstrate that host cell caveolae initiate the invasion process of group A streptococci and that the streptococcal invasin SfbI is the triggering factor that activates the caveolae-mediated endocytic pathway.

Microbial entry through caveolae: variations on a theme.

Caveolae and lipid rafts are increasingly being recognized as a significant portal of entry into host cells for a wide variety of pathogenic microorganisms. Entry through this mechanism appears to afford the microbes protection from degradation in lysosomes, though the level to which each microbe actively participates in avoiding lysosomal fusion may vary. Other possible variations in microbial entry through caveolae or lipid rafts may include (i) the destination of trafficking after entry and (ii) how actively the microbe contributes to the caveolae lipid/raft mediated entry. It seems that, though a wide variety of microorganisms are capable of utilizing caveolae/lipid rafts in various stages of their intracellular lifestyle, there can be distinct differences in how each microbe interacts with these structures. By studying these variations, we may learn more about the normal functioning of these cellular microdomains, and perhaps of more immediate importance, how to incorporate the use of these structures into the treatment of both infectious and non-infectious disease.

Caveolae in the uptake and targeting of infectious agents and secreted toxins.

A variety of microbial pathogens, including viruses, intracellular bacteria, and prions, as well as certain secreted bacterial toxins, can now be added to the list of ligands that enter cells via caveolae or caveolae-like membrane domains. In general, the caveolae-mediated entry pathway results in transport of these microbes and toxins to intracellular destinations that are different from that of cargo entering by other means. As a result, the caveolae-mediated entry pathway can profoundly affect the host cell-pathogen interaction long after entry has occurred. Furthermore, some microbes such as SV40 that enter via cavolae will be valuable as probes to analyze certain poorly understood intracellular trafficking pathways, such as retrograde transport to the ER. Also, viruses that enter via caveolae may have unique potential as gene and drug delivery vectors. In addition, some extracellular microbial pathogens, such as Pneumocystis carinii, may also interact with host cells via caveolae. Finally, caveolae may play a role in host immune defense mechanisms.

Caveolae as portals of entry for microbes.

Many pathogens, including many traditionally extracellular microbes, now appear capable of entry into host cells with limited loss of viability. A portal of entry shared by some bacteria, bacterial toxins, viruses and parasites are caveolae (or lipid rafts), which are involved in the import and intracellular translocation of macromolecules in host cells. A requirement for caveolae-mediated endocytosis of microbes appears to be that the respective receptor is a constituent of caveolae or must move to caveolae following ligation.

Glycosylphosphatidylinositol-anchored receptor-mediated bacterial endocytosis.

An increasing number of pathogens or their toxins appear to utilize glycosylphosphatidylinositol(GPI)-anchored receptors to trigger entry into immune and other host cells. Since these receptors have no transmembrane and intracellular moieties, how endocytosis is initiated is unclear. Recently, CD48 on mast cell membranes was shown to trigger endocytosis of bacteria via a route that avoids fusion with lysosomes and by a mechanism involving discrete cellular entities called caveolae. The localization of CD48 within caveolae appears to be a prerequisite for caveolae-mediated bacterial entry.

Co-option of endocytic functions of cellular caveolae by pathogens.

It is increasingly becoming clear that various immune cells are infected by the very pathogens that they are supposed to attack. Although many mechanisms for microbial entry exist, it appears that a common route of entry shared by certain bacteria, viruses and parasites involves cellular lipid-rich microdomains sometimes called caveolae. These cellular entities, which are characterized by their preferential accumulation of glycosylphosphatidylinositol (GPI)-anchored molecules, cholesterol and various glycolipids, and a distinct protein (caveolin), are present in many effector cells of the immune system including neutrophils, macrophages, mast cells and dendritic cells. These structures have an innate capacity to endocytoze various ligands and traffic them to different intracellular sites and sometimes, back to the extracellular cell surface. Because caveolae do not typically fuse with lysosomes, the ligands borne by caveolar vesicles are essentially intact, which is in marked contrast to ligands endocytozed via the classical endosome-lysosome pathway. A number of microbes or their exotoxins co-opt the unique features of caveolae to enter and traffic, without any apparent loss of viability and function, to different sites within immune and other host cells. In spite of their wide disparity in size and other structural attributes, we predict that a common feature among caveolae-utilizing pathogens and toxins is that their cognate receptor(s) are localized within plasmalemmal caveolae of the host cell.