Intestinal translocation of enterococci requires a threshold level of enterococcal overgrowth in the lumen

Enterococci are subdominant members of the human gastrointestinal microbiota. Enterococcus faecalis is generally harmless for healthy individuals, but it can cause a diverse range of infections in immunodeficient or elderly patients with severe underlying diseases. In this study, we analysed the levels of intestinal translocation of indigenous enterococci in C57BL/6, CF-1 and CX3CR1-/- mice upon clindamycin antibiotic-induced dysbiosis. We found that C57BL/6 was the most permissive model for enterococcal translocation and that initiation of E. faecalis translocation coincided with a threshold of enterococcal colonisation in the gut lumen, which once reached, triggered E. faecalis dissemination to deeper organs. We showed that the extent to which E. faecalis clinical strain VE14821 competed with indigenous enterococci differed between the C57BL/6 and CX3CR1-/- models. Finally, using a simplified gnotobiotic model, we observed E. faecalis crossing an intact intestinal tract using intestinal epithelial cells as one route to reach the lamina propria. Our study opens new perspectives for assessing the effect of various immunodeficiencies and for investigating mechanisms underlying enterococcal translocation

Visualization of the role of host heme on the virulence of the heme auxotroph Streptococcus agalactiae

International audienceHeme is essential for several cellular key functions but is also toxic. Whereas most bacterial pathogens utilize heme as a metabolic cofactor and iron source, the impact of host heme during bacterial infection remains elusive. The opportunist pathogen Streptococcus agalactiae does not synthesize heme but still uses it to activate a respiration metabolism. Concomitantly, heme toxicity is mainly controlled by the HrtBA efflux transporter. Here we investigate how S. agalactiae manages heme toxicity versus benefits in the living host. Using bioluminescent bacteria and heme-responsive reporters for in vivo imaging, we show that the capacity of S. agalactiae to overcome heme toxicity is required for successful infection, particularly in blood-rich organs. Host heme is simultaneously required, as visualized by a generalized infection defect of a respiration-negative mutant. In S. agalactiae, HrtBA expression responds to an intracellular heme signal via activation of the two-component system HssRS. A hssRS promoter-driven intracellular luminescent heme sensor was designed to identify host compartments that supply S. agalactiae with heme. S. agalactiae acquires heme in heart, kidneys, and liver, but not in the brain. We conclude that S. agalactiae response to heme is organ-dependent, and its efflux may be particularly relevant in late stages of infection

NAD+ pool depletion as a signal for the Rex regulon involved in Streptococcus agalactiae virulence

International audienceIn many Gram-positive bacteria, the redox-sensing transcriptional repressor Rex controls central carbon and energy metabolism by sensing the intra cellular balance between the reduced and oxidized forms of nicotinamide adenine dinucleotide; the NADH/NAD + ratio. Here, we report high-resolution crystal structures and characterization of a Rex ortholog (Gbs1167) in the opportunistic pathogen, Streptococcus agalactiae , also known as group B streptococcus (GBS). We present structures of Rex bound to NAD + and to a DNA operator which are the first structures of a Rex-family member from a pathogenic bacterium. The structures reveal the molecular basis of DNA binding and the conformation alterations between the free NAD + complex and DNA-bound form of Rex. Transcriptomic analysis revealed that GBS Rex controls not only central metabolism, but also expression of the monocistronic rex gene as well as virulence gene expression. Rex enhances GBS virulence after disseminated infection in mice. Mechanistically, NAD + stabilizes Rex as a repressor in the absence of NADH. However, GBS Rex is unique compared to Rex regulators previously characterized because of its sensing mechanism: we show that it primarily responds to NAD + levels (or growth rate) rather than to the NADH/NAD + ratio. These results indicate that Rex plays a key role in GBS pathogenicity by modulating virulence factor gene expression and carbon metabolism to harvest nutrients from the host

Nuclease A (Gbs0661), an extracellular nuclease of Streptococcus agalactiae, attacks the neutrophil extracellular traps and is needed for full virulence

Most bacteria of the genus Streptococcus are opportunistic pathogens, and some of them produce extracellular DNases, which may be important for virulence. Genome analyses of Streptococcus agalactiae (GBS) neonate isolate NEM316 revealed the presence of seven genes putatively encoding secreted DNases, although their functions, if any, are unknown. In this study, we observed that respiration growth of GBS led to the extracellular accumulation of a putative nuclease, identified as being encoded by the gbs0661 gene. When overproduced in Lactococcus lactis, the protein was found to be a divalent cation-requiring, pH-stable and heat-stable nuclease that we named Nuclease A (NucA). Substitution of the histidine(148) by alanine reduced nuclease activity of the GBS wild-type strain, indicating that NucA is the major nuclease ex vivo. We determined that GBS is able to degrade the DNA matrix comprising the neutrophil extracellular trap (NET). The nucA(H148A) mutant was impaired for this function, implicating NucA in the virulence of GBS. In vivo infection studies confirmed that NucA is required for full infection, as the mutant strain allowed increased bacterial clearance from lung tissue and decreased mortality in infected mice. These results show that NucA is involved in NET escape and is needed for full virulence