Selective inhibition of miRNA processing by a herpesvirus-encoded miRNA.

Herpesviruses have mastered host cell modulation and immune evasion to augment productive infection, life-long latency and reactivation1,2. A long appreciated, yet undefined relationship exists between the lytic-latent switch and viral non-coding RNAs3,4. Here we identify viral microRNA (miRNA)-mediated inhibition of host miRNA processing as a cellular mechanism that human herpesvirus 6A (HHV-6A) exploits to disrupt mitochondrial architecture, evade intrinsic host defences and drive the switch from latent to lytic virus infection. We demonstrate that virus-encoded miR-aU14 selectively inhibits the processing of multiple miR-30 family members by direct interaction with the respective primary (pri)-miRNA hairpin loops. Subsequent loss of miR-30 and activation of the miR-30-p53-DRP1 axis triggers a profound disruption of mitochondrial architecture. This impairs induction of type I interferons and is necessary for both productive infection and virus reactivation. Ectopic expression of miR-aU14 triggered virus reactivation from latency, identifying viral miR-aU14 as a readily druggable master regulator of the herpesvirus lytic-latent switch. Our results show that miRNA-mediated inhibition of miRNA processing represents a generalized cellular mechanism that can be exploited to selectively target individual members of miRNA families. We anticipate that targeting miR-aU14 will provide new therapeutic options for preventing herpesvirus reactivations in HHV-6-associated disorders.

Advanced human mucosal tissue models are needed to improve preclinical testing of vaccines.

Many vaccine candidates with promising results in preclinical testing fail in human trials. New complex human tissue models have the potential to improve the predictability of vaccine safety and efficacy in human clinical trials.

Intracellular Staphylococcus aureus employs the cysteine protease staphopain A to induce host cell death in epithelial cells.

Staphylococcus aureus is a major human pathogen, which can invade and survive in non-professional and professional phagocytes. Uptake by host cells is thought to contribute to pathogenicity and persistence of the bacterium. Upon internalization by epithelial cells, cytotoxic S. aureus strains can escape from the phagosome, replicate in the cytosol and induce host cell death. Here, we identified a staphylococcal cysteine protease to induce cell death after translocation of intracellular S. aureus into the host cell cytoplasm. We demonstrated that loss of staphopain A function leads to delayed onset of host cell death and prolonged intracellular replication of S. aureus in epithelial cells. Overexpression of staphopain A in a non-cytotoxic strain facilitated intracellular killing of the host cell even in the absence of detectable intracellular replication. Moreover, staphopain A contributed to efficient colonization of the lung in a mouse pneumonia model. In phagocytic cells, where intracellular S. aureus is exclusively localized in the phagosome, staphopain A did not contribute to cytotoxicity. Our study suggests that staphopain A is utilized by S. aureus to exit the epithelial host cell and thus contributes to tissue destruction and dissemination of infection.

Identification and initial characterization of a new pair of sibling sRNAs of Neisseria gonorrhoeae involved in type IV pilus biogenesis.

Non-coding regulatory RNAs mediate post-transcriptional gene expression control by a variety of mechanisms relying mostly on base-pairing interactions with a target mRNA. Though a plethora of putative non-coding regulatory RNAs have been identified by global transcriptome analysis, knowledge about riboregulation in the pathogenic Neisseriae is still limited. Here we report the initial characterization of a pair of sRNAs of N. gonorrhoeae, TfpR1 and TfpR2, which exhibit a similar secondary structure and identical single-stranded seed regions, and therefore might be considered as sibling sRNAs. By combination of in silico target prediction and sRNA pulse expression followed by differential RNA sequencing we identified target genes of TfpR1 which are involved in type IV pilus biogenesis and DNA damage repair. We provide evidence that members of the TfpR1 regulon can also be targeted by the sibling TfpR2.

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.

Chlamydia trachomatis impairs host base excision repair by downregulating polymerase ß.

Chlamydia trachomatis infections have been associated with ovarian cancer by several epidemiological studies. Here, we show that C. trachomatis-infected primary human ovarian epithelial cells display elevated oxidative DNA damage. Base excision repair, an important cellular mechanism to repair oxidative DNA lesions, was impaired in infected primary ovarian and in several other types of cells. Polymerase ß was downregulated in infected cells associated with upregulation of microRNA-499a (miR-499a). Stabilising polymerase ß by inhibiting miR-499a significantly improved repair. Moreover, downregulation of tumour suppressor p53 also resulted in attenuated repair in these cells. Thus, our data show that downregulation of polymerase ß by direct inhibition through miR-499a and downregulation of p53 debilitate the host-cell base excision repair during C. trachomatis infection.

Subversion of Cell-Autonomous Host Defense by Chlamydia Infection.

Obligate intracellular bacteria entirely depend on the metabolites of their host cell for survival and generation of progeny. Due to their lifestyle inside a eukaryotic cell and the lack of any extracellular niche, they have to perfectly adapt to compartmentalized intracellular environment of the host cell and counteract the numerous defense strategies intrinsically present in all eukaryotic cells. This so-called cell-autonomous defense is present in all cell types encountering Chlamydia infection and is in addition closely linked to the cellular innate immune defense of the mammalian host. Cell type and chlamydial species-restricted mechanisms point a long-term evolutionary adaptation that builds the basis of the currently observed host and cell-type tropism among different Chlamydia species. This review will summarize the current knowledge on the strategies pathogenic Chlamydia species have developed to subvert and overcome the multiple mechanisms by which eukaryotic cells defend themselves against intracellular pathogens.

Safe haven under constant attack-The Chlamydia-containing vacuole.

Chlamydia belong to the group of obligate intracellular bacteria that reside in a membrane bound vacuole during the entire intracellular phase of their life cycle. This vacuole called inclusion shields the bacteria from adverse influences in the cytosol of the host cell like the destructive machinery of the cell-autonomous defence system. The inclusion thereby prevents the digestion and eradication in specialised compartments of the intact and viable cell called phagolysosomes or autophagolysosomes. It is becoming more and more evident that keeping the inclusion intact also prevents the onset of cell intrinsic cell death programmes that are activated upon damage of the inclusion and direct the cell to destruct itself and the pathogen inside. Chlamydia secrete numerous proteins into the inclusion membrane to protect and stabilise their unique niche inside the host cell. We will focus in this review on the diverse attack strategies of the host aiming at the destruction of the Chlamydia-containing inclusion and will summarise the current knowledge on the protection mechanisms elaborated by the bacteria to maintain the integrity of their replication niche.

Natural mutations in a Staphylococcus aureus virulence regulator attenuate cytotoxicity but permit bacteremia and abscess formation.

Staphylococcus aureus is a major bacterial pathogen, which causes severe blood and tissue infections that frequently emerge by autoinfection with asymptomatically carried nose and skin populations. However, recent studies report that bloodstream isolates differ systematically from those found in the nose and skin, exhibiting reduced toxicity toward leukocytes. In two patients, an attenuated toxicity bloodstream infection evolved from an asymptomatically carried high-toxicity nasal strain by loss-of-function mutations in the gene encoding the transcription factor repressor of surface proteins (rsp). Here, we report that rsp knockout mutants lead to global transcriptional and proteomic reprofiling, and they exhibit the greatest signal in a genome-wide screen for genes influencing S. aureus survival in human cells. This effect is likely to be mediated in part via SSR42, a long-noncoding RNA. We show that rsp controls SSR42 expression, is induced by hydrogen peroxide, and is required for normal cytotoxicity and hemolytic activity. Rsp inactivation in laboratory- and bacteremia-derived mutants attenuates toxin production, but up-regulates other immune subversion proteins and reduces lethality during experimental infection. Crucially, inactivation of rsp preserves bacterial dissemination, because it affects neither formation of deep abscesses in mice nor survival in human blood. Thus, we have identified a spontaneously evolving, attenuated-cytotoxicity, nonhemolytic S. aureus phenotype, controlled by a pleiotropic transcriptional regulator/noncoding RNA virulence regulatory system, capable of causing S. aureus bloodstream infections. Such a phenotype could promote deep infection with limited early clinical manifestations, raising concerns that bacterial evolution within the human body may contribute to severe infection.

Long Noncoding RNA SSR42 Controls Staphylococcus aureus Alpha-Toxin Transcription in Response to Environmental Stimuli.

Staphylococcus aureus is a human pathogen causing a variety of diseases by versatile expression of a large set of virulence factors that most prominently features the cytotoxic and hemolytic pore-forming alpha-toxin. Expression of alpha-toxin is regulated by an intricate network of transcription factors. These include two-component systems sensing quorum and environmental signals as well as regulators reacting to the nutritional status of the pathogen. We previously identified the repressor of surface proteins (Rsp) as a virulence regulator. Acute cytotoxicity and hemolysis are strongly decreased in rsp mutants, which are characterized by decreased transcription of toxin genes as well as loss of transcription of a 1,232-nucleotide (nt)-long noncoding RNA (ncRNA), SSR42. Here, we show that SSR42 is the effector of Rsp in transcription regulation of the alpha-toxin gene, hla SSR42 transcription is enhanced after exposure of S. aureus to subinhibitory concentrations of oxacillin which thus leads to an SSR42-dependent increase in hemolysis. Aside from Rsp, SSR42 transcription is under the control of additional global regulators, such as CodY, AgrA, CcpE, and σB, but is positioned upstream of the two-component system SaeRS in the regulatory cascade leading to alpha-toxin production. Thus, alpha-toxin expression depends on two long ncRNAs, SSR42 and RNAIII, which control production of the cytolytic toxin on the transcriptional and translational levels, respectively, with SSR42 as an important regulator of SaeRS-dependent S. aureus toxin production in response to environmental and metabolic signals.IMPORTANCEStaphylococcus aureus is a major cause of life-threatening infections. The bacterium expresses alpha-toxin, a hemolysin and cytotoxin responsible for many of the pathologies of S. aureus Alpha-toxin production is enhanced by subinhibitory concentrations of antibiotics. Here, we show that this process is dependent on the long noncoding RNA, SSR42. Further, SSR42 itself is regulated by several global regulators, thereby integrating environmental and nutritional signals that modulate hemolysis of the pathogen.

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.

Staphylococcus aureus Exploits a Non-ribosomal Cyclic Dipeptide to Modulate Survival within Epithelial Cells and Phagocytes.

Community-acquired (CA) Staphylococcus aureus cause various diseases even in healthy individuals. Enhanced virulence of CA-strains is partly attributed to increased production of toxins such as phenol-soluble modulins (PSM). The pathogen is internalized efficiently by mammalian host cells and intracellular S. aureus has recently been shown to contribute to disease. Upon internalization, cytotoxic S. aureus strains can disrupt phagosomal membranes and kill host cells in a PSM-dependent manner. However, PSM are not sufficient for these processes. Here we screened for factors required for intracellular S. aureus virulence. We infected escape reporter host cells with strains from an established transposon mutant library and detected phagosomal escape rates using automated microscopy. We thereby, among other factors, identified a non-ribosomal peptide synthetase (NRPS) to be required for efficient phagosomal escape and intracellular survival of S. aureus as well as induction of host cell death. By genetic complementation as well as supplementation with the synthetic NRPS product, the cyclic dipeptide phevalin, wild-type phenotypes were restored. We further demonstrate that the NRPS is contributing to virulence in a mouse pneumonia model. Together, our data illustrate a hitherto unrecognized function of the S. aureus NRPS and its dipeptide product during S. aureus infection.

Inside job: Staphylococcus aureus host-pathogen interactions.

Staphylococcus aureus is a notorious opportunistic pathogen causing a plethora of diseases. Recent research established that once phagocytosed by neutrophils and macrophages, a certain percentage of S. aureus is able to survive within these phagocytes which thereby even may contribute to dissemination of the pathogen. S. aureus further induces its uptake by otherwise non-phagocytic cells and the ensuing intracellular cytotoxicity is suggested to lead to tissue destruction, whereas bacterial persistence within cells is thought to lead to immune evasion and chronicity of infections. We here review recent work on the S. aureus host pathogen interactions with a focus on the intracellular survival of the pathogen.

The role of host cell organelles in the development of Simkania negevensis.

Simkania negevensis is an obligate intracellular Chlamydia-like pathogen of the respiratory tract. It infects and multiplies in a wide range of hosts, from unicellular amoeba to a variety of human cells, such as epithelial HeLa and macrophage-like THP1 cells. The Simkania-containing vacuole (SnCV) forms close contacts with the endoplasmic reticulum (ER), and recruits and affects mitochondria of the host cells. Simkania prevent ER stress and require the components of the retrograde transport, as well as several mitochondrial and peroxisomal proteins, for proper development. This review recapitulates our current knowledge about the involvement of various cellular organelles in the life cycle of S. negevensis.

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 sibling sRNAs NgncR_162 and NgncR_163 of Neisseria gonorrhoeae participate in the expression control of metabolic, transport and regulatory proteins.

Neisseria gonorrhoeae is the causative agent of gonorrhoea, the second most common bacterial sexually transmitted disease. Riboregulation mediated by small regulatory RNAs (sRNAs) is increasingly recognized as an important means of gene expression control in this human-restricted pathogen. sRNAs act at the post-transcriptional level by base-pairing with their target mRNAs which affects translation initiation and/or mRNA stability. In this study we initiated the characterization of a pair of highly conserved sRNAs of N. gonorrhoeae which exhibit redundant functions in the control of a common set of target genes. The identified targets of the sibling sRNAs NgncR_162 and NgncR_163 participate in basic metabolic processes including the methylcitrate and citrate cycle, aa uptake and degradation, and also in transcription regulation. Our data indicate that the sibling sRNAs control their targets via direct base-pairing between the same single-stranded domain(s) of the sRNA and the ribosome binding site in the 5'-untranslated region of the mRNA.

Post-transcriptional regulation of target genes by the sRNA FnrS in Neisseria gonorrhoeae.

Small non-coding RNAs (sRNAs) are well-established post-transcriptional regulators of gene expression in bacteria that respond to a variety of environmental stimuli. They usually act by base-pairing with their target mRNAs, which is commonly facilitated by the RNA chaperone Hfq. In this study we initiated the analysis of the sRNA FnrS of Neisseria gonorrhoeae, which is induced under anaerobic conditions. We identified four putative FnrS target genes using bioinformatics approaches and validated these target genes using translational reporter gene fusions in both Escherichia coli and N. gonorrhoeae, thereby demonstrating their downregulation by direct base-pairing between the respective mRNA and FnrS. We demonstrate deregulation of target mRNAs upon deletion of fnrS and provide evidence that the isc gene cluster required for iron-sulfur cluster biosynthesis, which harbours iscS, which is a direct target of FnrS, is coordinately downregulated by the sRNA. By mutational analysis we show that, surprisingly, three distinct regions of FnrS are employed for interaction with different target genes.

EphrinA2 receptor (EphA2) is an invasion and intracellular signaling receptor for Chlamydia trachomatis.

The obligate intracellular bacterium Chlamydia trachomatis invades into host cells to replicate inside a membrane-bound vacuole called inclusion. Multiple different host proteins are recruited to the inclusion and are functionally modulated to support chlamydial development. Invaded and replicating Chlamydia induces a long-lasting activation of the PI3 kinase signaling pathway that is required for efficient replication. We identified the cell surface tyrosine kinase EphrinA2 receptor (EphA2) as a chlamydial adherence and invasion receptor that induces PI3 kinase (PI3K) activation, promoting chlamydial replication. Interfering with binding of C. trachomatis serovar L2 (Ctr) to EphA2, downregulation of EphA2 expression or inhibition of EphA2 activity significantly reduced Ctr infection. Ctr interacts with and activates EphA2 on the cell surface resulting in Ctr and receptor internalization. During chlamydial replication, EphA2 remains active accumulating around the inclusion and interacts with the p85 regulatory subunit of PI3K to support the activation of the PI3K/Akt signaling pathway that is required for normal chlamydial development. Overexpression of full length EphA2, but not the mutant form lacking the intracellular cytoplasmic domain, enhanced PI3K activation and Ctr infection. Despite the depletion of EphA2 from the cell surface, Ctr infection induces upregulation of EphA2 through the activation of the ERK pathway, which keeps the infected cell in an apoptosis-resistant state. The significance of EphA2 as an entry and intracellular signaling receptor was also observed with the urogenital C. trachomatis-serovar D. Our findings provide the first evidence for a host cell surface receptor that is exploited for invasion as well as for receptor-mediated intracellular signaling to facilitate chlamydial replication. In addition, the engagement of a cell surface receptor at the inclusion membrane is a new mechanism by which Chlamydia subverts the host cell and induces apoptosis resistance.

Chlamydia-infected cells shed Gp96 to prevent chlamydial re-infection.

Chlamydia trachomatis is an obligate intracellular human pathogen with a biphasic developmental life cycle. The infectious elementary bodies (EBs) enter a host cell where they transform into reticulate bodies (RBs) that use cellular metabolites to multiply. Re-infection of an infected cell during the replicative phase of chlamydial development may prevent formation of infectious EBs, interrupting the infectious cycle. Here, we report that Glucose Regulated Protein 96 (Gp96), a chaperone for cell surface receptors, binds to and facilitates adherence and entry of C. trachomatis. Gp96 expression was increased early in infection in a MAP kinase-dependent way, thereby increasing chlamydial adherence and invasion. Gp96 co-precipitated with Protein Disulphide Isomerase (PDI), known to be involved in chlamydial host cell entry. During the replicative phase, Gp96 was depleted from infected cells and shed into the supernatant by activation of metalloproteinase TACE (ADAM17). Loss of Gp96 also reduced the activity of PDI on the cell surface. Reduced surface display of Gp96 prevented chlamydial re-infection in a TACE-dependent manner in cell lines but also in primary cells derived from human fimbriae, the natural site of chlamydial infection. Our data suggest a role of infection-induced Gp96 shedding in the protection of the chlamydial replicative niche.