Biology subjects

Moldovan, A.

Publications and source records attributed to Moldovan, A..

4 recordsLinked to original sources

bioRxiv2024

Identification and characterization of a ubiquitin E3 RING ligase of the Chlamydia-like bacterium Simkania negevensis

In the arms race between a pathogen and the host, many bacteria have acquired a sizable armory to counteract or change the defense mechanisms of the host cell, including the eukaryotic ubiquitin system. Simkania negevensis, an obligate intracellular Chlamydia-like bacterium, is one such example. S. negevensis has a biphasic developmental cycle, similar to other members of the Chlamydiales order. The bacterium develops inside a tubular membranous compartment called Simkania-containing vacuole, which is in close contact with the host endoplasmic reticulum (ER) and mitochondria. It can infect a wide range of hosts, with a long infection cycle lasting up to 15 days, and is associated with respiratory tract diseases. Recently, S. negevensis has been discovered to possess an unusually large number of deubiquitinating enzymes, but not much is known about the variety of other ubiquitin-modifying enzymes in this bacterium. Our study provides an initial description of the activity of a so far uncharacterized S. negevensis ubiquitin E3 RING-ligase (SNE_A12920 or SneRING). We report that SneRING primarily generates K63- and K-11 linked ubiquitin chains and preferentially interacts with UbcH5b and UBE2T E2 ubiquitin ligases among the ones we tested. Bacteria express SneRING upon infection of various human cell lines, as well as amoeba. In addition, we demonstrate that a portion of the expressed SneRING co-localizes with mitochondria and ER. Mass spectrometry studies of the SneRING interactome show enrichment in mitochondrial and ER proteins containing a prohibitin domain and involved in organelle morphology, respiration, and stress response. Our work offers the first insights into the function of an S. negevensis RING ligase, an enzyme potentially involved in organelle remodeling to accommodate the unique lifestyle of this intracellular bacterium.

biochemistry↗

bioRxiv2024

A Novel Rapid Host Cell Entry Pathway Determines Intracellular Fate of Staphylococcus aureus

Staphylococcus aureus is an opportunistic pathogen causing severe diseases. Recently, S. aureus was recognized as intracellular pathogen, whereby the intracellular niche promotes immune evasion and antibiotic resistance. Interaction of S. aureus with versatile host cell receptors was described previously, suggesting that internalization of the pathogen can occur via several pathways. It remains elusive whether the pathway of internalization can affect the intracellular fate of the bacteria. Here, we identified a mechanism governing cellular uptake of S. aureus which relies on lysosomal Ca2+, lysosomal exocytosis and occurs concurrently to other well-known entry pathways within the same host cell population. This internalization pathway is rapid and active within only few minutes after bacterial contact with host cells. Compared to slow bacterial internalization, the rapid pathway demonstrates altered phagosomal maturation as well as translocation of the pathogen to the host cytosol and ultimately results in different rates of intracellular bacterial replication and host cell death. We show that these alternative infection outcomes are caused by the mode of bacterial uptake.

microbiology↗

bioRxiv2024

The AusAB non-ribosomal peptide synthase in Staphylococcus aureus preferentially incorporates exogenous phenylalanine and tyrosine into the aureusimine natural products

Non-ribosomal peptide synthases (NRPS) are modular multidomain enzymes, responsible for the biosynthesis of various secondary metabolites, in a mRNA-template independent manner. They are predominantly present in bacteria and fungi, where they synthesize a variety of products, including antibiotics, siderophores, toxins and signalling molecules. The human pathogen Staphylococcus aureus possesses one single NRPS, AusA, highly conserved in all sequenced S. aureus strains. AusA incorporates the aromatic amino acids (AAA) phenylalanine or tyrosine, as well as the branched-chain amino acids (BCAA) valine and leucine into three cyclic dipeptides collectively called aureusimines: phevalin, tyrvalin and leuvalin. By using targeted metabolomics, we found that AusA preferentially incorporates phenylalanine and tyrosine from an exogenous source into aureusimines, whereas the source of valine can be either endo- or exogenous. Upon cultivation in a chemically defined medium (CDM) lacking phenylalanine, the amino acid was not incorporated into phevalin, despite de novo phenylalanine biosynthesis. Tyrosine production remained unaffected. Conversely, upon cultivation in medium lacking tyrosine, tyrvalin production was not detected, despite tyrosine de novo biosynthesis. Phevalin production, however, remained unaltered. By contrast, omission of valine in the culture medium not only resulted in de novo valine biosynthesis but also was accompanied by phevalin production. To our knowledge, this is the first report of a selective incorporation of AAAs by a bacterial NRPS, which provides useful basis for linking bacterial cell metabolic status to the biosynthesis of secondary metabolites. IMPORTANCEPeptide and protein synthesis are fundamental processes in nature, which are largely mediated by the ribosomal machinery. An alternative pathway for peptide synthesis is non-ribosomal mRNA-template independent synthesis, performed by so-called non-ribosomal peptide synthases (NRPS). NRPSs are multi-enzyme complexes, which serve the simultaneous role of template and biosynthetic machinery. They are mostly found in bacteria and fungi and are responsible for the biosynthesis of many pharmacologically significant products, including antibiotics, anticancer compounds or immunosuppressants. The human pathogen S. aureus possesses one such NRPS, AusA, which synthesizes three cyclic dipeptides termed "aureusimines" using the aromatic amino acids phenylalanine and tyrosine, and the branched-chain amino acid valine. Although the biological role of aureusimines remains unknown, AusA appears to play a role in the interaction of S. aureus with the host. In addition, owing to its minimal canonical NRPS structure and autonomous function (i.e. most NRPS pathways require the assembly of several NRPS proteins), AusA represents an excellent model system for studying such molecular assembly lines. Our observation is, to our knowledge, the first report of a NRPS incorporating phenylalanine and tyrosine only from exogenous sources (e.g. environment, culture medium), but not from de novo "self-made" pool. This opens up new avenues in understanding and modulating the function of NRPSs (e.g. for biotechnological purposes).

microbiology↗

bioRxiv2020

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. Intracellularity 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 by intracellular S. aureus after translocation 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. Our study suggests that staphopain A is utilized by S. aureus to mediate escape from the host cell and thus contributes to tissue destruction and dissemination of infection. Author SummaryStaphylococcus aureus is a well-known antibiotic-resistant pathogen that emerges in hospital and community settings and can cause a variety of diseases ranging from skin abscesses to lung inflammation and blood poisoning. The bacterium asymptomatically colonizes the upper respiratory tract and skin of about one third of the human population and takes advantage of opportune conditions, like immunodeficiency or breached barriers, to cause infection. Although S. aureus is not regarded as a professional intracellular bacterium, it can be internalized by human cells and subsequently exit the host cells by induction of cell death, which is considered to cause tissue destruction and spread of infection. The bacterial virulence factors and underlying molecular mechanisms involved in the intracellular lifestyle of S. aureus remain largely unknown. We identified a bacterial cysteine protease to contribute to host cell death mediated by intracellular S. aureus. Staphopain A induced killing of the host cell after translocation of the pathogen into the cell cytosol, while bacterial proliferation was not required. Further, the protease enhanced survival of the pathogen during lung infection. These findings reveal a novel, intracellular role for the bacterial protease staphopain A.

microbiology↗