In vivo and in vitro analysis of the role of the Prc protease in inducing mucoidy in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, alginate biosynthesis gene expression is inhibited by the transmembrane anti-sigma factor MucA, which sequesters the AlgU sigma factor. Cell envelope stress initiates cleavage of the MucA periplasmic domain by site-1 protease AlgW, followed by further MucA degradation to release AlgU. However, after colonizing the lungs of people with cystic fibrosis, P. aeruginosa converts to a mucoid form that produces alginate constitutively. Mucoid isolates often have mucA mutations, with the most common being mucA22 , which truncates the periplasmic domain. MucA22 is degraded constitutively, and genetic studies suggested that the Prc protease is responsible. Some studies also suggested that Prc contributes to induction in strains with wild type MucA, whereas others suggested the opposite. However, missing from all previous studies is a demonstration that Prc cleaves any protein directly, which leaves open the possibility that the effect of a prc null mutation is indirect. To address the ambiguities and shortfalls, we reevaluated the roles of AlgW and Prc as MucA and MucA22 site-1 proteases. In vivo analyses using three different assays, and two different inducing conditions, all suggested that AlgW is the only site-1 protease for wild type MucA in any condition. In contrast, genetics suggested that AlgW or Prc act as MucA22 site-1 proteases in inducing conditions, whereas Prc is the only MucA22 site-1 protease in non-inducing conditions. For the first time, we also show that Prc is unable to degrade the periplasmic domain of wild type MucA, but does degrade the mutated periplasmic domain of MucA22 directly. IMPORTANCE: After colonizing the lungs of individuals with cystic fibrosis, P. aeruginosa undergoes mutagenic conversion to a mucoid form, worsening the prognosis. Most mucoid isolates have a truncated negative regulatory protein MucA, which leads to constitutive production of the extracellular polysaccharide alginate. The protease Prc has been implicated, but not shown, to degrade the most common MucA variant, MucA22, to trigger alginate production. This work provides the first demonstration that the molecular mechanism of Prc involvement is direct degradation of the MucA22 periplasmic domain, and perhaps other truncated MucA variants as well. MucA truncation and degradation by Prc might be the predominant mechanism of mucoid conversion in cystic fibrosis infections, suggesting that Prc activity could be a useful therapeutic target.

Capsular Polysaccharide Is Essential for the Virulence of the Antimicrobial-Resistant Pathogen Enterobacter hormaechei.

Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

Bacterial Carboxyl-Terminal Processing Proteases Play Critical Roles in the Cell Envelope and Beyond.

Proteolysis is essential throughout life, and as more proteases are characterized, our understanding of the roles they play continues to expand. Among other things, proteases are critical for protein turnover and quality control, the activation or inactivation of some enzymes, and they are integral components of signal transduction pathways. This review focuses on a family of proteases in bacteria known as the carboxyl-terminal processing proteases, or CTPs. Members of this family occur in all domains of life. In bacteria, CTPs have emerged as important enzymes that have been implicated in critical processes including regulation, stress response, peptidoglycan remodeling, and virulence. Here, we provide an overview of the roles that CTPs play in diverse bacterial species, and some of the underlying mechanisms. We also describe the structures of some bacterial CTPs, and their adaptor proteins, which have revealed striking differences in arrangements and mechanisms of action. Finally, we discuss what little is known about the distinguishing features of CTP substrates and cleavage sites, and speculate about how CTP activities might be regulated in the bacterial cell. Compared with many other proteases, the study of bacterial CTPs is still in its infancy, but it has now become clear that they affect fundamental processes in many different species. This is a protease family with broad significance, and one that holds the promise of more high impact discoveries to come.