Legionella pneumophila, an environmental bacterium that parasitizes protozoa, is the causative pathogen of Legionnaires' disease. L. pneumophila adopts a distinct biphasic life cycle that allows it to adapt to environmental conditions for survival, replication, and transmission. This cycle consists of a non-virulent replicative phase (RP) and a virulent transmissive phase (TP). Timely and fine-tuned expression of growth and virulence factors in a life cycle-dependent manner is crucial. Herein, we report evidence that CsrA, a key regulator of the switch between the RP and the TP, is dually regulated in a ClpP-dependent manner during the biphasic life cycle of L. pneumophila. First, we show that the protein level of CsrA is temporal during the life cycle and is degraded by ClpP during the TP. The ectopic expression of CsrA in a ΔclpP mutant, but not in the wild type, inhibits both the initiation of the RP in vitro and the invasiveness to Acanthamoeba castellanii, indicating that the ClpP-mediated proteolytic pathway regulates the CsrA protein level. We further show that the temporally expressed IHFB is the transcriptional inhibitor of csrA and is degraded via a ClpP-dependent manner during the RP. During the RP, the level of CsrA is increased by promoting the degradation of IHFB and reducing the degradation of the accumulated CsrA via a ClpP-dependent manner. During the TP, the level of CsrA is decreased by inhibiting the degradation of IHFB and promoting the degradation of the accumulated CsrA via a ClpP-dependent manner as well. In conclusion, our results show that the growth-stage-specific expression level of CsrA is dually regulated by ClpP-dependent proteolysis at both the transcription and protein levels during the biphasic life cycle of L. pneumophila.
Secreted proteins are one of the direct molecular mechanisms by which microbiota influence the host, thus constituting a promising field for drug discovery. Here, through bioinformatics-guided screening of the secretome of clinically established probiotics from Lactobacillus, we identify an uncharacterized secreted protein (named LPH here) that is shared by most of these probiotic strains (8/10) and demonstrate that it protects female mice from colitis in multiple models. Functional studies show that LPH is a bi-functional peptidoglycan hydrolase with both N-Acetyl-β-D-muramidase and DL-endopeptidase activities that can generate muramyl dipeptide (MDP), a NOD2 ligand. Different active site mutants of LPH in combination with Nod2 knockout female mice confirm that LPH exerts anti-colitis effects through MDP-NOD2 signaling. Furthermore, we validate that LPH can also exert protective effects on inflammation-associated colorectal cancer in female mice. Our study reports a probiotic enzyme that enhances NOD2 signaling in vivo in female mice and describes a molecular mechanism that may contribute to the effects of traditional Lactobacillus probiotics.
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