Tissue Culture Models Of Infection Research Articles

Human GCIP interacts with CT847, a novel Chlamydia trachomatis type III secretion substrate, and is degraded in a tissue-culture infection model

The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole and employs a type III secretion mechanism to translocate host-interactive proteins. These proteins most likely contribute to pathogenesis through modulation of host cell mechanisms crucial for the establishment and maintenance of a permissive intracellular environment. Using a surrogate Yersinia type III secretion system (T3SS), we have identified the conserved gene product CT847 as a chlamydial T3SS substrate. Yeast two-hybrid studies using CT847 as bait to screen a HeLa cell cDNA library identified an interaction with mammalian Grap2 cyclin D-interacting protein (GCIP). Immunoblot analyses of C. trachomatis-infected HeLa cells showed that GCIP levels begin to decrease (as compared with mock-infected HeLa cells) between 8 h and 12 h post infection. GCIP was virtually undetectable in 24 h time point material. This decrease was inhibited by proteasome inhibitors lactacystin and MG-132, and the T3SS inhibitor Compound 1. CT847 was detectible in purified reticulate body but not elementary body lysates, and reverse transcription polymerase chain reaction (RT-PCR) expression analyses indicate a mid-cycle expression pattern. Both of these findings are consistent with CT847 contributing to the observed effect on GCIP. Given the established roles of GCIP, we believe that we have discovered a novel C. trachomatis antihost protein whose activity is relevant to chlamydial pathogenesis.

Helicobacter pylori CagA Induces AGS Cell Elongation through a Cell Retraction Defect That Is Independent of Cdc42, Rac1, and Arp2/3

Helicobacter pylori, which infects over one-half the world's population, is a significant risk factor in a spectrum of gastric diseases, including peptic ulcers and gastric cancer. Strains of H. pylori that deliver the effector molecule CagA into host cells via a type IV secretion system are associated with more severe disease outcomes. In a tissue culture model of infection, CagA delivery results in a dramatic cellular elongation referred to as the "hummingbird" phenotype, which is characterized by long, thin cellular extensions. These actin-based cytoskeletal rearrangements are reminiscent of structures that are regulated by Rho GTPases and the Arp2/3 complex. We tested whether these signaling pathways were important in the H. pylori-induced cell elongation phenotype. Contrary to our expectations, we found that these molecules are dispensable for cell elongation. Instead, time-lapse video microscopy revealed that cells infected by cagA(+) H. pylori become elongated because they fail to release their back ends during cell locomotion. Consistent with a model in which CagA causes cell elongation by inhibiting the disassembly of adhesive cell contacts at migrating cells' lagging ends, immunohistochemical analysis revealed that focal adhesion complexes persist at the distal tips of elongated cell projections. Thus, our data implicate a set of signaling molecules in the hummingbird phenotype that are different than the molecules previously suspected.

Multinucleated Giant Cell Formation and Apoptosis in Infected Host Cells Is Mediated by Burkholderia pseudomallei Type III Secretion Protein BipB

Here we have assessed the role of a type III translocator protein, BipB, in the cell biology and virulence of Burkholderia pseudomallei. Genetic inactivation of bipB reduced multinucleated giant cell formation, cell-to-cell spreading of bacteria, and induction of apoptosis of J774A.1 macrophages. The bipB mutant was also significantly attenuated following intranasal challenge of BALB/c mice, whereas virulence was fully restored by complementation with a functional bipB gene. Burkholderia pseudomallei, the etiological agent of melioidosis in humans and animals, is a gram-negative bacterium. Melioidosis is endemic in southeast Asia and tropical Australia and has been reported sporadically elsewhere (6). Currently, there is no vaccine against melioidosis. Uniquely among intracellular bacterial pathogens, B. pseudomallei induces host cell fusion leading to multinucleated giant cell (MNGC) formation in tissue culture models of infection (14). This novel phenotype may be relevant to pathogenesis, since granuloma formation and generation of MNGC are also found in tissues of humans with melioidosis (23). In addition to inducing MNGC formation, B. pseudomallei is able to spread from cell to cell and induce apoptotic death in infected host cells (14). The molecular mechanisms of these pathogenic characteristics have not been elucidated. Analysis of the B. pseudomallei genome and several other studies have demonstrated the presence of a type III secretion system (TTSS) (for reviews, see references 3, 12, 17, 20, and 22). A knockout mutant of B. pseudomallei lacking a functional bipD gene, a homologue of Salmonella enterica serovar Typhimurium sipD, on the TTSS3/bsa cluster of TTSS exhibited reduced replication in murine macrophage-like cells (20), was significantly attenuated in BALB/c mice and gave partial protection against subsequent challenge with wild-type B. pseudomallei (19). These data correlated with the recent report that the TTSS3/bsa cluster is required for the pathogenicity of B. pseudomallei (21). In addition to BipD, B. pseudomallei BipB and BipC (46 and 30% amino acid identity to Salmonella SipB and SipC, respectively) have been identified in the TTSS3/bsa cluster (3). Here, we report on the role of BipB in the pathogenesis of infection with B. pseudomallei. With Salmonella organisms, purified SipB integrates into artificial membranes and induces liposome fusion (10), and it is required for inducing apoptosis in murine macrophages (11). By analogy with SipB, therefore, we investigated the role of BipB for MNGC formation, cell-to-cell spreading, and induction of apoptosis in infected host cells. We also examined the virulence of a B. pseudomallei bipB mutant in a murine model of melioidosis.

Temporal Expression of Enteropathogenic Escherichia coli Virulence Genes in an In Vitro Model of Infection

The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the ability of EPEC to cause attaching and effacing (A/E) lesions on intestinal epithelium. This event is reproducible in in vitro tissue culture models of infection. We used real-time PCR to measure transcription from several locus of enterocyte effacement (LEE) operons (LEE1 to LEE5) and from bfp during a 5-h infection of HEp-2 cells with EPEC. We found that after the initial formation of A/E lesions, which occurs as early as 5 min postinfection, EPEC continues to increase transcription from LEE3 to LEE5 as well as from bfp. These levels are maximized by 3 h postinfection and remain constant throughout the course of infection. This increase in transcription from LEE3 to LEE5 occurs when LEE1 (ler) transcription is decreasing. EspA, EspB, intimin, Tir, and bundle-forming pilus expression is detectable during the entire 5-h infection. These results indicate that the EPEC genes involved in localized and intimate adherence are continually expressed after the initial stages of A/E lesion formation on host cells.

A polymerase chain reaction-based protocol for the detection of transmission of pig endogenous retroviruses in pig to human xenotransplantation.

Xenotransplantation of pig organs and tissues to humans bears the risk of infection of immunosuppressed recipients by porcine endogenous retrovirus (PERV) released from the transplanted tissue. However, when diagnosing potential PERV transmission, it is essential to exclude microchimerism, i.e., persisting pig cells in analyzed bioptic material of xenotransplanted patients, which give rise to false positive PERV signals. Polymerase chain reaction (PCR) is so far the only suitable method to diagnose a cross-species transfer of PERV, but the exclusion of microchimerism might be a serious problem because most of the presently employed primer pairs detect PERV sequences with higher sensitivity than primers used for the detection of contaminating pig sequences. We designed and evaluated a novel and improved primer set for detection of pig sequences as well as complementing positive control primers on the basis of mitochondrial cytochrome B, an approved marker for phylogenetic studies. We further established primer pairs derived from the long terminal repeat/leader region of PERV isolated from a Duroc German Landrace cross-bred pig and tested their sensitivity in comparison with known PERV- and pig-specific PCR markers. In standard PCR assays, the new cytochrome B-derived primers are at least 10 times more sensitive than the presently used PERV retroviral polymerase gene and mammalian beta-actin primers. When tested in a tissue culture infection model, PERV transmission to human 293 cells can be unambiguously demonstrated, even in the presence of up to 10% pig cells. One of the primer combinations derived from the PERV DuxDL3791 long terminal repeat/leader region amplifies with even lower sensitivity than primers detecting porcine beta-globin, thus permitting the exclusion of microchimerism also via chromosomal loci. The availability of the new PCR markers allows the proposal of a rigorous setup for the routine detection of PERV transmission after xenotransplantation.

Preliminary characterization of a Mycobacterium abscessus mutant in human and murine models of infection.

The ability to persist in the host after the establishment of infection is an important virulence determinant for mycobacteria. Mycobacterium abscessus is a rapidly growing mycobacterial species which causes a variety of clinical syndromes in humans. We have obtained a rough, wild-type human clinical isolate of M. abscessus (M. abscessus-R) and a smooth, attenuated mutant (M. abscessus-S) which spontaneously dissociated from the clinical isolate. We have found that M. abscessus-R is able to persist and multiply in a murine pulmonary infection model in contrast to M. abscessus-S, which is rapidly cleared. To understand the basis for this difference, we characterized the behavior of these variants in human tissue culture models of infection. M. abscessus-R is able to persist and multiply in human monocytes, while M. abscessus-S is deficient in this ability. Both of these variants are phagocytized by human monocytes. M. abscessus-R resides in a phagosome typical for pathogenic mycobacteria with a tightly adherent phagosomal membrane. In contrast, M. abscessus-S resides in a "loose" phagosome with the phagosomal membrane separated from the bacterial cell wall. Both M. abscessus variants also have distinctive growth patterns in a recently described fibroblast-mycobacterium microcolony assay, with M. abscessus-R exhibiting growth characteristics similar to those previously reported for virulent M. tuberculosis and M. abscessus-S exhibiting growth characteristics similar to those previously reported for avirulent M. tuberculosis. In both the monocyte infection assay and the murine pulmonary infection model, numerous infected mononuclear phagocyte aggregates develop at sites of M. abscessus-R infection, but are absent with M. abscessus-S infection. We conclude that a mutation has occurred in the M. abscessus-S variant which has altered the ability of this organism to persist and multiply in host cells and that this may be related to the phenotypic changes we have observed in our tissue culture models of infection.

LcrV of Yersinia pestis enters infected eukaryotic cells by a virulence plasmid-independent mechanism.

Yersinia pestis is the causative agent of bubonic plague and possesses a set of plasmid-encoded, secretable virulence proteins termed LcrV and Yops which are essential for survival in mammalian hosts. Yops and LcrV are secreted by a type III mechanism (Ysc), and Yops are unidirectionally targeted into the cytosol of associated eukaryotic cells in a tissue culture infection model. LcrV is required for Yops targeting, and recent findings have revealed that it can localize to the bacterial surface; however, its fate in this infection model has not been investigated in detail. In this study, we compared the localization of LcrV to that of the targeted proteins YopE and YopM by immunoblot analysis of fractions of Yersinia-infected HeLa cultures or by laser-scanning confocal microscopy of infected monolayers. Both LcrV and YopE were secreted by contact-activated, extracellularly localized yersiniae and were targeted to the HeLa cell cytosol. Although a significant amount of LcrV partitioned to the culture medium (unlike YopE), this extracellular pool of LcrV was not the source of the LcrV that entered HeLa cells. Unlike targeting of YopE and YopM, targeting of LcrV occurred in the absence of a functional Ysc apparatus and other virulence plasmid (pCD1)-expressed proteins. However, the Ysc is necessary for LcrV to be released into the medium, and our recent work has shown that localization of LcrV on the bacterial surface requires the Ysc. These results indicate that two mechanisms exist for the secretion of LcrV by Y. pestis, both of which are activated by contact with eukaryotic cells. LcrV secreted by the Ysc reaches the bacterial surface and the surrounding medium, whereas the second is a novel, Ysc-independent pathway which results in localization of LcrV in the cytosol of infected cells but not the surrounding medium.

Dual promoters of the Listeria monocytogenes prfA transcriptional activator appear essential in vitro but are redundant in vivo

The PrfA transcriptional activator is an essential determinant of Listeria monocytogenes pathogenesis. prfA expression is governed by three differentially regulated promoters: prfAP1 and prfAP2, which are located immediately upstream of prfA coding sequences, and the adjacent plcA promoter via the generation of a plcA-prfA read-through transcript. A series of promoter deletion mutants were constructed to assess the roles of prfAP1 and prfAP2. Elimination of either prfAP1 or prfAP2 resulted in altered regulation of PrfA-regulated genes after in vitro growth. However, these mutants were fully virulent both in an animal model and in tissue culture models of infection, suggesting that the two prfA promoters are functionally redundant in vivo. In contrast, a mutant lacking both prfAP1 and prfAP2 was 100-fold less virulent and was delayed in escape from the host vacuole. Once in the host cytoplasm, however, the double mutant was apparently normal in cell-to-cell spread.