The effects of medium and rate of freezing on the survival of chlamydias after lyophilization

The effects of suspension media and rate of freezing on the survival of Chlamydia trachomatis LGV₂ and Chlamydia pneumoniae after lyophilization were assessed. The highest loss in infectious elementary bodies (EBs) occurred during lyophilization. The survival was higher after freezing at a rate of 1°C min^-1 and lyophilization than that after rapid freezing at - 70°C or - 196°C. The recovery (± 5%) was higher when fetal calf serum (FCS) containing glucose, saccharose or lactose were used as lyophilization media than that (0.5–3%) when yolk-sac, skimmed milk or phosphate buffer containing sucrose, glutamine and 10% FCS (SPG) were used. After lyophilization, the survival was not affected in the tested range from 10⁴ to 5 times 10⁶ inclusion-forming units (ifu) ml^-1 prior to freezing. After storage for 4 months at 4°C, the numbers of ifu of both Chlamydia serovars that were recovered were identical to the numbers of ifu immediately after lyophilization. It was concluded that chlamydias can be stored and transported in lyophilized form. However, a loss of 95% in infectious EBs should be taken into account.     

Host cell-derived sphingolipids are required for the intracellular growth of Chlamydia trachomatis

Chlamydia trachomatis , an important cause of human disease, is an obligate intracellular bacterial pathogen that relies on the eukaryotic host cell for its replication. Recent reports have revealed that the C. trachomatis vacuole receives host-derived sphingolipids by fusing with trans -Golgi network (TGN)-derived secretory vesicles. Here, it is shown that these lipids are required for the growth of the bacteria. C. trachomatis was unable to replicate at 39°C in the Chinese hamster ovary (CHO)-derived cell line SPB-1, a cell line incapable of synthesizing sphingolipids at this temperature because of a temperature-sensitive mutation in the serine palmitoyltransferase (SPT) gene. Complementation with the wild-type SPT gene or addition of exogenous cell-permeable sphingolipid precursors to the mutant cells restored their ability to support chlamydial replication. l -cycloserine ( l -CS) and fumonisin B₁ (FB1), inhibitors of sphingolipid biosynthesis, decreased the proliferation of the bacteria in eukaryotic cells at concentrations that also decreased host cell sphingolipid synthesis. In the case of FB₁, the vacuoles appeared aberrant; the addition of sphingolipid precursors was able to reverse the altered morphology of the FB₁-treated vacuoles. Collectively, these data strongly suggest that the growth and replication of chlamydiae is dependent on synthesis of sphingolipids by the eukaryotic host cell and may contribute to this organism's obligate intracellular parasitism.     

Determination of chlamydial load and immune parameters in asymptomatic, symptomatic and infertile women

The regulation of immune response and chlamydial infectious load in the cervix of human females is largely unknown. Infectious load in terms of inclusion-forming units (IFUs) was determined by quantitative cultures in Chlamydia -positive women, in asymptomatic women, women with mucopurulent cervicitis (MPC) and women with fertility disorders (FD). CD4⁺, CD8⁺, CD14⁺ cells, myeloid and plasmacytoid dendritic cells (mDCs and pDCs) in the cervix were quantified by flow cytometry. Cervical cytokines, levels of β-estradiol and C-reactive protein (CRP) in serum and cervical immunoglobulin A antibody to chlamydial major outer membrane protein antigen, chlamydial heat shock protein 60 and 10 antigens were measured by an enzyme-linked immunosorbent assay. In asymptomatic women, chlamydial load showed significant positive correlations with CD4, mDCs, interleukin-12 (IL-12) and IL-2; however, negative correlations were found with CD8 and IL-8 levels. In women with MPC, chlamydial IFUs correlated positively with CD8, pDC number, IL-8, CRP and interferon-γ (IFN-γ). In women with FD, chlamydial load showed a significant positive correlation with the pDC number, IL-10 and estradiol level and a negative correlation with CD4 and IFN-γ. Overall, these results suggest that the interplay between chlamydial infectious load and host immune responses may be the deciding factor for the clinical condition presented during Chlamydia trachomatis infection.     

The chlamydial deubiquitinase Cdu1 supports recruitment of Golgi vesicles to the inclusion

Chlamydia trachomatis is the main cause of sexually transmitted diseases worldwide. As obligate intracellular bacteria Chlamydia replicate in a membrane bound vacuole called inclusion and acquire nutrients for growth and replication from their host cells. However, like all intracellular bacteria, Chlamydia have to prevent eradication by the host's cell autonomous system. The chlamydial deubiquitinase Cdu1 is secreted into the inclusion membrane, facing the host cell cytosol where it deubiquitinates cellular proteins. Here we show that inactivation of Cdu1 causes a growth defect of C. trachomatis in primary cells. Moreover, ubiquitin and several autophagy receptors are recruited to the inclusion membrane of Cdu1‐deficient Chlamydia. Interestingly, the growth defect of cdu1 mutants is not rescued when autophagy is prevented. We find reduced recruitment of Golgi vesicles to the inclusion of Cdu1 mutants indicating that vesicular trafficking is altered in bacteria without active deubiquitinase (DUB). Our work elucidates an important role of Cdu1 in the functional preservation of the chlamydial inclusion surface.     

Cytochrome c-mediated caspase-9 activation triggers apoptosis in Streptococcus pyogenes-infected epithelial cells

Epithelial cells are the initial sites of host invasion by group A Streptococcus pyogenes (GAS), and their infection of epithelial cells has been suggested to induce apoptosis. However, the mechanism responsible for bacteria–host interaction and the induction of apoptosis has not been clearly understood. We demonstrate here that human pharyngeal epithelial HEp-2 cells became apoptotic with DNA fragmentation by invasion of GAS strains JRS4 (M6⁺, F1⁺) and JRS145 (M6⁻, F1⁺ mutant of JRS4), whereas apoptotic cellular changes were not observed in SAM1 (M6⁺, F1⁻ mutant) or SAM2 (M6⁻, F1⁻ mutant) infected HEp-2 cells. Confocal microscopy revealed that Bax translocation to mitochondria and cytochrome c release occurred after 4 h of infection. Western blot analyses showed that the amounts of Bcl-2 and Bcl-x_L were decreased in the mitochondria of infected cells. In addition, we demonstrated that the release of nuclear histone from infected cells was prevented by the addition of caspase-9 inhibitor (Ac-LEHD-CHO). We conclude that the internalization of GAS in epithelial cells is necessary and sufficient for the induction of apoptosis, which is initiated by mitochondrial dysfunction, and the mechanism of GAS-induced apoptosis is clearly different from that induced by other intracellular invasive bacteria, e.g. Shigella and Salmonella species.     

Hidden Behind Autophagy: The Unconventional Roles of ATG Proteins

Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved intracellular catabolic transport route that generally allows the lysosomal degradation of cytoplasmic components, including bulk cytosol, protein aggregates, damaged or superfluous organelles and invading microbes. Target structures are sequestered by double‐membrane vesicles called autophagosomes, which are formed through the concerted action of the autophagy (ATG)‐related proteins. Until recently it was assumed that ATG proteins were exclusively involved in autophagy. A growing number of studies, however, have attributed functions to some of them that are distinct from their classical role in autophagosome biogenesis. Autophagy‐independent roles of the ATG proteins include the maintenance of cellular homeostasis and resistance to pathogens. For example, they assist and enhance the turnover of dead cells and microbes upon their phagocytic engulfment, and inhibit murine norovirus replication. Moreover, bone resorption by osteoclasts, innate immune regulation triggered by cytoplasmic DNA and the ER‐associated degradation regulation all have in common the requirement of a subset of ATG proteins. Microorganisms such as coronaviruses, Chlamydia trachomatis or Brucella abortus have even evolved ways to manipulate autophagy‐independent functions of ATG proteins in order to ensure the completion of their intracellular life cycle. Taken together these novel mechanisms add to the repertoire of functions and extend the number of cellular processes involving the ATG proteins.     

Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.     

An early event in the herpes simplex virus type-2 replication cycle is sufficient to induce Chlamydia trachomatis persistence

Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virus-induced chlamydial persistence was neither host cell- nor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development.     

Crohn's disease-associated adherent-invasive E. coli are selectively favoured by impaired autophagy to replicate intracellularly

Ileal lesions in Crohn's disease (CD) patients are colonized by pathogenic adherent-invasive Escherichia coli (AIEC) able to invade and to replicate within intestinal epithelial cells. Recent genome-wide association studies have highlighted the autophagy pathway as being associated with CD risk. In the present study we investigated whether defects in autophagy enhance replication of commensal and pathogenic Escherichia coli and CD-associated AIEC. We show that functional autophagy limits intracellular AIEC replication and that a subpopulation of the intracellular bacteria is located within LC3-positive autophagosomes. In IRGM and ATG16L1 deficient cells intracellular AIEC LF82 bacteria have enhanced replication. Surprisingly autophagy deficiency did not interfere with the ability of intracellular bacteria to survive and/or replicate for any other E. coli strains tested, including non-pathogenic, environmental, commensal, or pathogenic strains involved in gastro enteritis. Together these findings demonstrate a central role for autophagy restraining Adherent-Invasive E. coli strains associated with ileal CD. AIEC infection in patients with polymorphisms in autophagy genes may have a significant impact on the outcome of intestinal inflammation.     

Crucial role of the local micro-environment in fate decision of neonatal rat NG2 progenitors

Objectives:  The fate choice of neural progenitor cells could be dictated by local cellular environment of the adult CNS. The aim of our study was to investigate the effect of hippocampal tissue on differentiation and maturation of oligodendrocyte NG2 precursor cells.
Materials and methods:  Hippocampal slice culture was established from the brains of 7-day-old rats. NG2 precursor cells, obtained from a 12-day-old mixed primary culture of neonatal rat cerebral hemispheres, were labelled with chloromethyl-fluorescein-diacetete and seeded on the hippocampal slices. After 7–14 days in co-culture, cells were stained with neural markers.
Results:  NG2 cells differentiated predominantly into oligodendrocytes, presenting various stages of maturation: progenitors (NG2), pre-oligodendrocytes (O4) and finally mature GalC-positive cells. However, except for a few cells with astrocyte-specific S100b staining, a considerable number of these cells differentiated into neurons: TUJ⁺ and even MAP-2⁺ cells were frequently observed. Moreover, a certain population of these cells preserved proliferative properties of primary precursor cells, as revealed by Ki67 expression.
Conclusions:  The neuronal micro-environment provided by the culture of hippocampal slices is potent for induction of neurogenesis from oligodendrocyte NG2⁺/PDGFRα⁺/CNP⁺ progenitor cells and promotes their differentiation not only into macroglia but also into neurons. It also sustains their proliferative capacity. The results indicate the crucial role of the local cellular environment in fate decision of primary NG2⁺ multipotent neural progenitor cells, which may affect their behaviour after transplantation into the central nervous system.     

Chlamydial infection induces host cytokinesis failure at abscission

Chlamydia trachomatis is an obligate intracellular bacteria and the infectious agent responsible for the sexually transmitted disease Chlamydia. Infection with Chlamydia can lead to serious health sequelae such as pelvic inflammatory disease and reproductive tract scarring contributing to infertility and ectopic pregnancies. Additionally, chlamydial infections have been epidemiologically linked to cervical cancer in patients with a prior human papilomavirus (HPV) infection. Chlamydial infection of cultured cells causes multinucleation, a potential pathway for chromosomal instability. Two mechanisms that are known to initiate multinucleation are cell fusion and cytokinesis failure. This study demonstrates that multinucleation of the host cell by Chlamydia is entirely due to cytokinesis failure. Moreover, cytokinesis failure is due in part to the chlamydial effector CPAF acting as an anaphase promoting complex mimic causing cells to exit mitosis with unaligned and unattached chromosomes. These lagging and missegregated chromosomes inhibit cytokinesis by blocking abscission, the final stage of cytokinesis.     

Francisella tularensis Harvests Nutrients Derived via ATG5-Independent Autophagy to Support Intracellular Growth

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.     

Unique glycine-activated riboswitch linked to glycine–serine auxotrophy in SAR11

The genome sequence of the marine bacterium ' Candidatus Pelagibacter ubique' and subsequent analyses have shown that while it has a genome as small as many obligate parasites, it nonetheless possesses a metabolic repertoire that allows it to grow as one of the most successful free-living cells in the ocean. An early report based on metabolic reconstruction indicated that SAR11 cells are prototrophs for all amino acids. However, here we report experimental evidence that ' Cand. P. ubique' is effectively auxotrophic for glycine and serine. With glucose and acetate added to seawater to supply organic carbon, the addition of 125 nM to 1.5 μM glycine to growth medium containing all other nutrients in excess resulted in a linear increase in maximum cell density from 1.14 × 10⁶ cells ml⁻¹ to 8.16 × 10⁶ cells ml⁻¹ ( R ² =  0.992). Serine was capable of substituting for glycine at 1.5 μM. ' Cand. P. ubique' contains a glycine-activated riboswitch preceding malate synthase, an unusual genomic context that is conserved in the SAR11 group. Malate synthase plays a critical role in central metabolism by enabling TCA intermediates to be regenerated through the glyoxylate cycle. In vitro analysis of this riboswitch indicated that it responds solely to glycine but not close structural analogues, such as glycine betaine, malate, glyoxylate, glycolate, alanine, serine or threonine. We conclude that ' Cand. P. ubique' is therefore a glycine–serine auxotroph that appears to use intracellular glycine level to regulate its use of carbon for biosynthesis and energy. Comparative genomics and metagenomics indicate that these conclusions may hold throughout much of the SAR11 clade.     

The Chlamydia pneumoniae Invasin Protein Pmp21 Recruits the EGF Receptor for Host Cell Entry

Infection of mammalian cells by the strictly intracellular pathogens Chlamydiae requires adhesion and internalization of the infectious Elementary Bodies (EBs). The components of the latter step were unknown. Here, we identify Chlamydia pneumoniae Pmp21 as an invasin and EGFR as its receptor. Modulation of EGFR surface expression evokes correlated changes in EB adhesion, internalization and infectivity. Ectopic expression of EGFR in EGFR-negative hamster cells leads to binding of Pmp21 beads and EBs, thus boosting the infection. EB/Pmp21 binding and invasion of epithelial cells results in activation of EGFR, recruitment of adaptors Grb2 and c-Cbl and activation of ERK1/2, while inhibition of EGFR or MEK kinase activity abrogates EB entry, but not attachment. Binding of Grb2 and c-Cbl by EGFR is essential for infection. This is the first report of an invasin-receptor interaction involved in host-cell invasion by any chlamydial species.     

High-yield culture and purification of Chlamydiaceae bacteria

Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.