Streptococcus agalactiae is a common human commensal and a major life-threatening pathogen in neonates. Adherence to host epithelial cells is the first critical step of the infectious process. Pili have been observed on the surface of several gram-positive bacteria including S. agalactiae. We previously characterized the pilus-encoding operon gbs1479-1474 in strain NEM316. This pilus is composed of three structural subunit proteins: Gbs1478 (PilA), Gbs1477 (PilB), and Gbs1474 (PilC), and its assembly involves two class C sortases (SrtC3 and SrtC4). PilB, the bona fide pilin, is the major component; PilA, the pilus associated adhesin, and PilC, are both accessory proteins incorporated into the pilus backbone. We first addressed the role of the housekeeping sortase A in pilus biogenesis and showed that it is essential for the covalent anchoring of the pilus fiber to the peptidoglycan. We next aimed at understanding the role of the pilus fiber in bacterial adherence and at resolving the paradox of an adhesive but dispensable pilus. Combining immunoblotting and electron microscopy analyses, we showed that the PilB fiber is essential for efficient PilA display on the surface of the capsulated strain NEM316. We then demonstrated that pilus integrity becomes critical for adherence to respiratory epithelial cells under flow-conditions mimicking an in vivo situation and revealing the limitations of the commonly used static adherence model. Interestingly, PilA exhibits a von Willebrand adhesion domain (VWA) found in many extracellular eucaryotic proteins. We show here that the VWA domain of PilA is essential for its adhesive function, demonstrating for the first time the functionality of a prokaryotic VWA homolog. Furthermore, the auto aggregative phenotype of NEM316 observed in standing liquid culture was strongly reduced in all three individual pilus mutants. S. agalactiae strain NEM316 was able to form biofilm in microtiter plate and, strikingly, the PilA and PilB mutants were strongly impaired in biofilm formation. Surprisingly, the VWA domain involved in adherence to epithelial cells was not required for biofilm formation.

Key Points• GATA1 is downregulated in RPS19-deficient cells and zebrafish through upregulation of p53, TNF-a, and p38 MAPK.• Treatment of rps19-deficient zebrafish with the TNF-a inhibitor etanercept rescues their erythroid and developmental defects.Diamond-Blackfan anemia (DBA) is an inherited disorder characterized by defects in erythropoiesis, congenital abnormalities, and predisposition to cancer. Approximately 25% of DBA patients have a mutation in RPS19, which encodes a component of the 40S ribosomal subunit. Upregulation of p53 contributes to the pathogenesis of DBA, but the link between ribosomal protein mutations and erythropoietic defects is not well understood. We found that RPS19 deficiency in hematopoietic progenitor cells leads to decreased GATA1 expression in the erythroid progenitor population and p53-dependent upregulation of tumor necrosis factor-a (TNF-a) in nonerythroid cells. The decrease in GATA1 expression was mediated, at least in part, by activation of p38 MAPK in erythroid cells and rescued by inhibition of TNF-a or p53. The anemia phenotype in rps19-deficient zebrafish was reversed by treatment with the TNF-a inhibitor etanercept. Our data reveal that RPS19 deficiency leads to inflammation, p53-dependent increase in TNF-a, activation of p38 MAPK, and decreased GATA1 expression, suggesting a novel mechanism for the erythroid defects observed in DBA. (Blood. 2014;124(25):3791-3798)

Cutibacterium acnes (former Propionibacterium acnes ), is a bacterium characterized by high genomic variability, consisting of four subtypes and six major ribotypes. Skin is the largest neuroendocrine organ of the human body and many cutaneous hormones and neurohormones can modulate b acterial physiology. Here, we investigated the effect of catecholamines, i.e., epinephrine and norepinephrine, on two representative strains of C. acnes , of which the genome has been fully sequenced, identified as RT4 acneic and RT6 non-acneic strains. Epinephrine and norepinephrine (10 −6 M) had no impact on the growth of C. acnes but epinephrine increased RT4 and RT6 biofilm formation, as measured by crystal violet staining, whereas norepinephrine was only active on the RT4 strain. We obtained the same results by confocal microscopy with the RT4 strain, whereas there was no effect of either catecholamine on the RT6 strain. However, this strain was also sensitive to catecholamines, as shown by MATs tests, as epinephrine and norepinephrine affected its surface polarity. Flow cytometry studies revealed that epinephrine and norepinephrine are unable to induce major changes of bacterial surface properties and membrane integrity. Exposure of sebocytes to control or catecholamine-treated bacteria showed epinephrine and norepinephrine to have no effect on the cytotoxic or inflammatory potential of either C. acnes strains but to stimulate their effect on sebocyte lipid synthesis. Uriage thermal spring water was previously shown to inhibit biofilm production by C. acnes . We thus tested its effect after exposure of the bacteria to epinephrine and norepinephrine. The effect of the thermal water on the response of C. acnes to catecholamines depended on the surface on which the biofilm was grown. Finally, an in-silico study revealed the presence of a protein in the genome of C. acnes that shows homology with the catecholamine receptor of Escherichia coli and eukaryotes. This study suggests that C. acnes may play a role as a relay between stress mediators (catecholamines) and acne.

Staphylococcus aureus and Staphylococcus epidermidis are two major skin associated bacteria, and Substance P (SP) is a major skin neuropeptide. Since bacteria are known to sense and response to many human hormones, we investigated the effects of SP on Staphylococci virulence in reconstructed human epidermis model and HaCaT keratinocytes. We show that SP is stimulating the virulence of S. aureus and S. epidermidis in a reconstructed human epidermis model. qRT-PCR array analysis of 64 genes expressed by keratinocytes in the response to bacterial infection revealed a potential link between the action of SP on Staphylococci and skin physiopathology. qRT-PCR and direct assay of cathelicidin and human β-defensin 2 secretion also provided that demonstration that the action of SP on bacteria is independent of antimicrobial peptide expression by keratinocytes. Considering an effect of SP on S. aureus and S. epidermidis, we observed that SP increases the adhesion potential of both bacteria on keratinocytes. However, SP modulates the virulence of S. aureus and S. epidermidis through different mechanisms. The response of S. aureus is associated with an increase in Staphylococcal Enterotoxin C2 (SEC2) production and a reduction of exolipase processing whereas in S. epidermidis the effect of SP appears mediated by a rise in biofilm formation activity. The Thermo unstable ribosomal Elongation factor Ef-Tu was identified as the SP-interacting protein in S. aureus and S. epidermidis. SP appears as an inter-kingdom communication factor involved in the regulation of bacterial virulence and essential for skin microflora homeostasis.

The biocontrol agent Rhodococcus erythropolis disrupts virulence of plant and human Gram-negative pathogens by catabolizing their N-acyl-homoserine lactones. This quorum-quenching activity requires the expression of the qsd (quorum-sensing signal degradation) operon, which encodes the lactonase QsdA and the fatty acyl-CoA ligase QsdC, involved in the catabolism of lactone ring and acyl chain moieties of signaling molecules, respectively. Here, we demonstrate the regulation of qsd operon expression by a TetR-like family repressor, QsdR. This repression was lifted by adding the pathogen quorum signal or by deleting the qsdR gene, resulting in enhanced lactone degrading activity. Using interactomic approaches and transcriptional fusion strategy, the qsd operon derepression was elucidated: it is operated by the binding of the common part of signaling molecules, the homoserine lactone ring, to the effector-receiving domain of QsdR, preventing a physical binding of QsdR to the qsd promoter region. To our knowledge, this is the first evidence revealing quorum signals as inducers of the suitable quorum-quenching pathway, confirming this TetR-like protein as a lactone sensor. This regulatory mechanism designates the qsd operon as encoding a global disrupting pathway for degrading a wide range of signal substrates, allowing a broad spectrum anti-virulence activity mediated by the rhodococcal biocontrol agent. Understanding the regulation mechanisms of qsd operon expression led also to the development of biosensors useful to monitor in situ the presence of exogenous signals and quorum-quenching activity.

Staphylococci can sense Substance P (SP) in skin, but this molecule is generally released by nerve terminals along with another neuropeptide, Calcitonin Gene Related Peptide (CGRP). In this study, we investigated the effects of αCGRP on Staphylococci. CGRP induced a strong stimulation of Staphylococcus epidermidis virulence with a low threshold (<10−12 M) whereas Staphylococcus aureus was insensitive to CGRP. We observed that CGRP-treated S. epidermidis induced interleukin 8 release by keratinocytes. This effect was associated with an increase in cathelicidin LL37 secretion. S. epidermidis displayed no change in virulence factors secretion but showed marked differences in surface properties. After exposure to CGRP, the adherence of S. epidermidis to keratinocytes increased, whereas its internalization and biofilm formation activity were reduced. These effects were correlated with an increase in surface hydrophobicity. The DnaK chaperone was identified as the S. epidermidis CGRP-binding protein. We further showed that the effects of CGRP were blocked by gadolinium chloride (GdCl3), an inhibitor of MscL mechanosensitive channels. In addition, GdCl3 inhibited the membrane translocation of EfTu, the Substance P sensor. This work reveals that through interaction with specific sensors S. epidermidis integrates different skin signals and consequently adapts its virulence.

The pioneering work of Kaplan and Greenberg [1] led to admit that, as eukaryotic cells, bacteria can communicate. In fact, many multicellular social bacterial behaviours such as swarming type motility, [2] biofilm formation [3] and virulence expression, [4] require population synchronization and that is performed at least partly through a highly regulated cell-to-cell communication system called quorum sensing (QS). QS is based on the bacterial population density, which is performed through secretion and sensing of specific signal molecules named autoinducers. Nowadays, many QS autoinducers, such as the N-acyl homoserine lactones (AHL) and quinolones (Gram-negative

Flagella-driven motility is an important trait for bacterial colonization and virulence. Flagella rotate and propel bacteria in liquid or semi-liquid media to ensure such bacterial fitness. Bacterial flagella are composed of three parts: a membrane complex, a flexible-hook, and a flagellin filament. The most widely studied models in terms of the flagellar apparatus are E. coli and Salmonella. However, there are many differences between these enteric bacteria and the bacteria of the Pseudomonas genus. Enteric bacteria possess peritrichous flagella, in contrast to Pseudomonads, which possess polar flagella. In addition, flagellar gene expression in Pseudomonas is under a four-tiered regulatory circuit, whereas enteric bacteria express flagellar genes in a three-step manner. Here, we use knowledge of E. coli and Salmonella flagella to describe the general properties of flagella and then focus on the specificities of Pseudomonas flagella. After a description of flagellar structure, which is highly conserved among Gram-negative bacteria, we focus on the steps of flagellar assembly that differ between enteric and polar-flagellated bacteria. In addition, we summarize generalities concerning the fuel used for the production and rotation of the flagellar macromolecular complex. The last part summarizes known regulatory pathways and potential links with the type-six secretion system (T6SS).