Metabolic adaptation of Chlamydia trachomatis to mammalian host cells.

Metabolic adaptation is a key feature for the virulence of pathogenic intracellular bacteria. Nevertheless, little is known about the pathways in adapting the bacterial metabolism to multiple carbon sources available from the host cell. To analyze the metabolic adaptation of the obligate intracellular human pathogen Chlamydia trachomatis, we labeled infected HeLa or Caco-2 cells with 13 C-marked glucose, glutamine, malate or a mix of amino acids as tracers. Comparative GC-MS-based isotopologue analysis of protein-derived amino acids from the host cell and the bacterial fraction showed that C. trachomatis efficiently imported amino acids from the host cell for protein biosynthesis. FT-ICR-MS analyses also demonstrated that label from exogenous 13 C-glucose was efficiently shuffled into chlamydial lipopolysaccharide probably via glucose 6-phosphate of the host cell. Minor fractions of bacterial Ala, Asp, and Glu were made de novo probably using dicarboxylates from the citrate cycle of the host cell. Indeed, exogenous 13 C-malate was efficiently taken up by C. trachomatis and metabolized into fumarate and succinate when the bacteria were kept in axenic medium containing the malate tracer. Together, the data indicate co-substrate usage of intracellular C. trachomatis in a stream-lined bipartite metabolism with host cell-supplied amino acids for protein biosynthesis, host cell-provided glucose 6-phosphate for cell wall biosynthesis, and, to some extent, one or more host cell-derived dicarboxylates, e.g. malate, feeding the partial TCA cycle of the bacterium. The latter flux could also support the biosynthesis of meso-2,6-diaminopimelate required for the formation of chlamydial peptidoglycan.

Proteomic analysis of the Simkania-containing vacuole: the central role of retrograde transport.

Simkania negevensis is an obligate intracellular bacterial pathogen that grows in amoeba or human cells within a membrane-bound vacuole forming endoplasmic reticulum (ER) contact sites. The membrane of this Simkania-containing vacuole (SnCV) is a critical host-pathogen interface whose origin and molecular interactions with cellular organelles remain poorly defined. We performed proteomic analysis of purified ER-SnCV-membranes using label free LC-MS(2) to define the pathogen-containing organelle composition. Of the 1,178 proteins of human and 302 proteins of Simkania origin identified by this strategy, 51 host cell proteins were enriched or depleted by infection and 57 proteins were associated with host endosomal transport pathways. Chemical inhibitors that selectively interfere with trafficking at the early endosome-to-trans-Golgi network (TGN) interface (retrograde transport) affected SnCV formation, morphology and lipid transport. Our data demonstrate that Simkania exploits early endosome-to-TGN transport for nutrient acquisition and growth.

The chlamydial organism Simkania negevensis forms ER vacuole contact sites and inhibits ER-stress.

Most intracellular bacterial pathogens reside within membrane-surrounded host-derived vacuoles. Few of these bacteria exploit membranes from the host's endoplasmic reticulum (ER) to form a replicative vacuole. Here, we describe the formation of ER-vacuole contact sites as part of the replicative niche of the chlamydial organism Simkania negevensis. Formation of ER-vacuole contact sites is evolutionary conserved in the distantly related protozoan host Acanthamoeba castellanii. Simkania growth is accompanied by mitochondria associating with the Simkania-containing vacuole (SCV). Super-resolution microscopy as well as 3D reconstruction from electron micrographs of serial ultra-thin sections revealed a single vacuolar system forming extensive ER-SCV contact sites on the Simkania vacuolar surface. Simkania infection induced an ER-stress response, which was later downregulated. Induction of ER-stress with Thapsigargin or Tunicamycin was strongly inhibited in cells infected with Simkania. Inhibition of ER-stress was required for inclusion formation and efficient growth, demonstrating a role of ER-stress in the control of Simkania infection. Thus, Simkania forms extensive ER-SCV contact sites in host species evolutionary as diverse as human and amoeba. Moreover, Simkania is the first bacterial pathogen described to interfere with ER-stress induced signalling to promote infection.

Modulation of host signaling and cellular responses by Chlamydia.

Modulation of host cell signaling and cellular functions is key to intracellular survival of pathogenic bacteria. Intracellular growth has several advantages e.g. escape from the humoral immune response and access to a stable nutrient rich environment. Growth in such a preferred niche comes at the price of an ongoing competition between the bacteria and the host as well as other microbes that compete for the very same host resources. This requires specialization and constant evolution of dedicated systems for adhesion, invasion and accommodation. Interestingly, obligate intracellular bacteria of the order Chlamydiales have evolved an impressive degree of control over several important host cell functions. In this review we summarize how Chlamydia controls its host cell with a special focus on signal transduction and cellular modulation.

Imbalanced oxidative stress causes chlamydial persistence during non-productive human herpes virus co-infection.

Both human herpes viruses and Chlamydia are highly prevalent in the human population and are detected together in different human disorders. Here, we demonstrate that co-infection with human herpes virus 6 (HHV6) interferes with the developmental cycle of C. trachomatis and induces persistence. Induction of chlamydial persistence by HHV6 is independent of productive virus infection, but requires the interaction and uptake of the virus by the host cell. On the other hand, viral uptake is strongly promoted under co-infection conditions. Host cell glutathione reductase activity was suppressed by HHV6 causing NADPH accumulation, decreased formation of reduced glutathione and increased oxidative stress. Prevention of oxidative stress restored infectivity of Chlamydia after HHV6-induced persistence. We show that co-infection with Herpes simplex virus 1 or human Cytomegalovirus also induces chlamydial persistence by a similar mechanism suggesting that Chlamydia -human herpes virus co-infections are evolutionary shaped interactions with a thus far unrecognized broad significance.

Evolutionary conservation of infection-induced cell death inhibition among Chlamydiales.

Control of host cell death is of paramount importance for the survival and replication of obligate intracellular bacteria. Among these, human pathogenic Chlamydia induces the inhibition of apoptosis in a variety of different host cells by directly interfering with cell death signaling. However, the evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. Here, we investigated the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway. Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales.

Tarp regulates early Chlamydia-induced host cell survival through interactions with the human adaptor protein SHC1.

Many bacterial pathogens translocate effector proteins into host cells to manipulate host cell functions. Here, we used a protein microarray comprising virtually all human SRC homology 2 (SH2) and phosphotyrosine binding domains to comprehensively and quantitatively assess interactions between host cell proteins and the early phase Chlamydia trachomatis effector protein translocated actin-recruiting phosphoprotein (Tarp), which is rapidly tyrosine phosphorylated upon host cell entry. We discovered numerous novel interactions between human SH2 domains and phosphopeptides derived from Tarp. The adaptor protein SHC1 was among Tarp's strongest interaction partners. Transcriptome analysis of SHC1-dependent gene regulation during infection indicated that SHC1 regulates apoptosis- and growth-related genes. SHC1 knockdown sensitized infected host cells to tumor necrosis factor-induced apoptosis. Collectively, our findings reveal a critical role for SHC1 in early C. trachomatis-induced cell survival and suggest that Tarp functions as a multivalent phosphorylation-dependent signaling hub that is important during the early phase of chlamydial infection.

Complex kinase requirements for Chlamydia trachomatis Tarp phosphorylation.

Chlamydia trachomatis translocates the effector protein Tarp (translocated actin-recruiting phosphoprotein) into the host cell cytoplasm where it is quickly tyrosine phosphorylated. Abl and Src kinases have been implicated in Tarp phosphorylation; however, we observed that the situation is more complex. Chemical inhibition of Src family kinases confirmed a role for these kinases in Tarp phosphorylation. Infection of Src, Yes, Fyn (SYF)-deficient cells showed a dampened, but incompletely blocked, Tarp phosphorylation. Inhibition of Abl in an SYF background still did not completely block Tarp phosphorylation. Consequently, we tested additional kinases and found that Syk, but not Btk or Jak2, is a potent kinase of Tarp in vitro. Inhibition of Syk in an SYF background further blocked Tarp phosphorylation. Under these conditions, inclusion formation still proceeded normally. These data reveal a highly promiscuous substrate property of Tarp and set the stage for further functional characterization of Tarp phosphorylation during host cell infection.

Gene expression profiles of Chlamydophila pneumoniae during the developmental cycle and iron depletion-mediated persistence.

The obligate intracellular, gram-negative bacterium Chlamydophila pneumoniae (Cpn) has impact as a human pathogen. Little is known about changes in the Cpn transcriptome during its biphasic developmental cycle (the acute infection) and persistence. The latter stage has been linked to chronic diseases. To analyze Cpn CWL029 gene expression, we designed a pathogen-specific oligo microarray and optimized the extraction method for pathogen RNA. Throughout the acute infection, ratio expression profiles for each gene were generated using 48 h post infection as a reference. Based on these profiles, significantly expressed genes were separated into 12 expression clusters using self-organizing map clustering and manual sorting into the "early", "mid", "late", and "tardy" cluster classes. The latter two were differentiated because the "tardy" class showed steadily increasing expression at the end of the cycle. The transcriptome of the Cpn elementary body (EB) and published EB proteomics data were compared to the cluster profile of the acute infection. We found an intriguing association between "late" genes and genes coding for EB proteins, whereas "tardy" genes were mainly associated with genes coding for EB mRNA. It has been published that iron depletion leads to Cpn persistence. We compared the gene expression profiles during iron depletion-mediated persistence with the expression clusters of the acute infection. This led to the finding that establishment of iron depletion-mediated persistence is more likely a mid-cycle arrest in development rather than a completely distinct gene expression pattern. Here, we describe the Cpn transcriptome during the acute infection, differentiating "late" genes, which correlate to EB proteins, and "tardy" genes, which lead to EB mRNA. Expression profiles during iron mediated-persistence led us to propose the hypothesis that the transcriptomic "clock" is arrested during acute mid-cycle.