title: LytM proteins play a crucial role in cell separation, outer membrane composition, and pathogenesis in nontypeable Haemophilus influenzae.
abstract: LytM proteins belong to a family of bacterial metalloproteases. In Gram-negative bacteria, LytM factors are mainly reported to have a direct effect on cell division by influencing cleavage and remodeling of peptidoglycan. In this study, mining nontypeable Haemophilus influenzae (NTHI) genomes, three highly conserved open reading frames (ORFs) containing a LytM domain were identified, and the proteins encoded by the ORFs were named YebA, EnvC, and NlpD on the basis of their homology with the Escherichia coli proteins. Immunoblotting and confocal analysis showed that while NTHI NlpD is exposed on the bacterial surface, YebA and EnvC reside in the periplasm. NTHI ΔyebA and ΔnlpD deletion mutants revealed an aberrant division phenotype characterized by an altered cell architecture and extensive membrane blebbing. The morphology of the ΔenvC deletion mutant was identical to that of the wild-type strain, but it showed a drastic reduction of periplasmic proteins, including the chaperones HtrA, SurA, and Skp, and an accumulation of β-barrel-containing outer membrane proteins comprising the autotransporters Hap, IgA serine protease, and HMW2A, as observed by proteomic analysis. These data suggest that EnvC may influence the bacterial surface protein repertoire by facilitating the passage of the periplasmic chaperones through the peptidoglycan layer to the close vicinity of the inner face of the outer membrane. This hypothesis was further corroborated by the fact that an NTHI envC defective strain had an impaired capacity to adhere to epithelial cells and to form biofilm. Notably, this strain also showed a reduced serum resistance. These results suggest that LytM factors are not only important components of cell division but they may also influence NTHI physiology and pathogenesis by affecting membrane composition.
  
  
  OBJECTIVE
  Nontypeable Haemophilus influenzae (NTHI) is an opportunistic pathogen that colonizes the human nasopharynx and can cause serious infections in children (acute otitis media) and adults (chronic obstructive pulmonary disease). Several virulence factors are well studied, but the complete scenario of NTHI pathogenesis is still unclear. We identified and characterized three NTHI LytM factors homologous to the Escherichia coli LytM proteins. Although LytM factors are reported to play a crucial role in the cell division process, in NTHI they are also involved in other bacterial functions. In particular, YebA and NlpD are fundamental for membrane stability: indeed, their absence causes an increased release of outer membrane vesicles (OMVs). On the other hand, our data suggest that EnvC could directly or indirectly affect peptidoglycan permeability and consequently, bacterial periplasmic and outer membrane protein distribution. Interestingly, by modulating the surface composition of virulence determinants, EnvC also has an impact on NTHI pathogenesis.
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Ercoli
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Tani
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Alfredo
authors.initials: A
authors.lastname: Pezzicoli
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Irene
authors.initials: I
authors.lastname: Vacca
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Manuele
authors.initials: M
authors.lastname: Martinelli
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Simone
authors.initials: S
authors.lastname: Pecetta
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Petracca
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Rino
authors.initials: R
authors.lastname: Rappuoli
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Mariagrazia
authors.initials: M
authors.lastname: Pizza
authors.affiliation: Novartis Vaccines, Siena, Italy nathalie.norais@novartis.com beatrice.arico@novartis.com.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Norais
authors.affiliation: Novartis Vaccines, Siena, Italy.
authors.forename: Marco
authors.initials: M
authors.lastname: Soriani
authors.affiliation: Novartis Vaccines, Siena, Italy nathalie.norais@novartis.com beatrice.arico@novartis.com.
authors.forename: Beatrice
authors.initials: B
authors.lastname: Aricò
mesh_terms: D001422:Bacterial Adhesion; D002455:Cell Division; D002462:Cell Membrane / Q000737:chemistry*; D002473:Cell Wall / Q000737:chemistry; D002478:Cells, Cultured; D019295:Computational Biology; D004847:Epithelial Cells / Q000382:microbiology; D017353:Gene Deletion; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D045726:Metalloproteases / Q000378:metabolism*; D016366:Open Reading Frames; D019897:Periplasm / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014774:Virulence; D037521:Virulence Factors
chemical_list: D037521:Virulence Factors; D045726:Metalloproteases