in-vitro-reconstitution-bacterial-ubiquitination-vcpp97-mediated

<em>In-vitro</em> Reconstitution of Bacterial Ubiquitination and VCP/p97-mediated Elimination

Ubiquitination is a versatile post-translational modification that plays a critical role in cytosolic immunity. Upon invasion of the host cells, ...

Indian Institute of Technology Bombay

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCF E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3β, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Cell-autonomous immunity prevents intracellular pathogen growth through mechanisms such as ubiquitination and proteasomal targeting of bacteria for degradation. However, how the proteasome eradicates ubiquitinated bacteria has remained unclear. Here we show that host AAA-ATPase, VCP/p97, associates with diverse cytosol-exposed ubiquitinated bacteria (Streptococcus pneumoniae, Salmonella enterica serovar Typhimurium, Streptococcus pyogenes) and requires the ATPase activity in its D2 domain to reduce intracellular bacterial loads. Combining optical trap approaches along with molecular dynamic simulations, in vitro reconstitution and immunogold transmission electron microscopy, we demonstrate that p97 applies mechanical force to extract ubiquitinated surface proteins, BgaA and PspA, from S. pneumoniae cell membranes. This causes extensive membrane lysis and release of cytosolic content, thereby killing the pathogen. Further, p97 also controls S. pneumoniae proliferation in mice, ultimately protecting from fatal sepsis. Overall, we discovered a distinct innate antimicrobial function of p97 that can protect the host against lethal bacterial infections.

Host AAA-ATPase VCP/p97 lyses ubiquitinated intracellular bacteria as an innate antimicrobial defence.

Opportunistic pathogens switch from a commensal to pathogenic state by sensing and responding to a variety of environmental cues, including temperature fluctuations. Minor temperature oscillations can alert the pathogen to a changing niche ecosystem, necessitating efficient sensing and rapid integration to trigger behavioral change. This is typically achieved through master regulators, dictating pleiotropic phenotypes. Here, we uncover a pivotal role of minor temperature shifts in transition of Streptococcus pneumoniae (SPN) from commensal to virulent lifestyles, mediated via an RNA thermosensing (RNAT) element within the untranslated region of the global regulator CiaR. By positively regulating the expression of the surface adhesin, Phosphorylcholine (PCho), in response to elevated temperature, CiaR potentiates pneumococcal infection. Engineering the RNAT structure to create translation restrictive or permissive versions allowed us to demonstrate how modulation in expression of CiaR could alter pneumococcal invasion capability, influencing infection outcomes. Moreover, intranasal administration of PCho mitigated SPN-induced bacteraemic pneumonia. Since a majority of opportunistic respiratory bacterial pathogens decorate their surface with PCho, this signaling arm could be exploited for anti-infective interventions.

Article	Year
An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins. Science advances\| PubMed ID: 36947610	2023
Host AAA-ATPase VCP/p97 lyses ubiquitinated intracellular bacteria as an innate antimicrobial defence. Nature microbiology\| PubMed ID: 40217128	2025
Commensal to pathogen switch in Streptococcus pneumoniae is influenced by a thermosensing master regulator. PLoS pathogens\| PubMed ID: 41026799	2025

Sourav Ghosh

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