Nitrite Reductase NirS Is Required for Type III Secretion System Expression and Virulence in the Human Monocyte Cell Line THP-1 by Pseudomonas aeruginosa

Abstract

The nitrate dissimilation pathway is important for anaerobic growth in Pseudomonas aeruginosa. In addition, this pathway contributes to P. aeruginosa virulence by using the nematode Caenorhabditis elegans as a model host, as well as biofilm formation and motility. We used a set of nitrate dissimilation pathway mutants to evaluate the virulence of P. aeruginosa PA14 in a model of P. aeruginosa-phagocyte interaction by using the human monocytic cell line THP-1. Both membrane nitrate reductase and nitrite reductase enzyme complexes were important for cytotoxicity during the interaction of P. aeruginosa PA14 with THP-1 cells. Furthermore, deletion mutations in genes encoding membrane nitrate reductase (⌬narGH) and nitrite reductase (⌬nirS) produced defects in the expression of type III secretion system (T3SS) components, extracellular protease, and elastase. Interestingly, exotoxin A expression was unaffected in these mutants. Addition of exogenous nitric oxide (NO)-generating compounds to ⌬nirS mutant cultures restored the production of T3SS phospholipase ExoU, whereas nitrite addition had no effect. These data suggest that NO generated via nitrite reductase NirS contributes to the regulation of expression of selected virulence factors in P. aeruginosa PA14.The ubiquitous gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen responsible for both acute and chronic infections. P. aeruginosa is commonly an etiologic agent in ear infections (8), infections of burn wounds (27), corneal keratitis (21, 30), and pulmonary infections in patients with cystic fibrosis (16) and ventilator-associated pneumonia (5). P. aeruginosa is frequently resistant to conventional antibiotic therapy and the antimicrobial effector mechanisms of phagocytes (9), particularly in the biofilm mode of growth (15).The establishment of P. aeruginosa infection is accompanied by the synthesis of a diverse array of virulence factors composed of various exoproteins, such as elastase and exotoxin A, as well as the mucoid exopolysaccharide alginate (20). The type III secretion system (T3SS), a mechanism whereby cytotoxic effector proteins are directly secreted into the host cell cytoplasm following contact of the bacterium with a target cell (11), has also been identified as a virulence determinant of P. aeruginosa that contributes significantly to the pathogenesis of acute infection (45, 46). The P. aeruginosa T3SS plays a major role in triggering cell death in phagocytes and epithelial cells (17) and is composed of at least 20 proteins, including (i) a secretory apparatus, (ii) machinery devoted to the direct translocation of effectors into the host cell cytoplasm, and (iii) four effector proteins, ExoS, ExoT, ExoU, and ExoY, that contribute to host cell toxicity (19,46). Regulation of virulence factor expression in P. aeruginosa is hierarchical and highly complex, involving quorum sensing (37, 44) and responses to environmental signals, including stresses applied via host defense mechanisms (46). P. aeruginosa is capable of growin...

Anr and Its Activation by PlcH Activity in Pseudomonas aeruginosa Host Colonization and Virulence

“…Induction of denitrification enzymes when oxygen is present may enable the detoxification of nitric oxide species produced in the highly inflammatory host environment (61). Small quantities of nitrate are available to be metabolized by P. aeruginosa in the CF lung (52,62,63), and nitric oxide, either provided exogenously or generated through denitrification, could stimulate the expression of the type III secretion system, which is associated with acute toxicity to epithelial cells and promotion of inflammation (64). The early induction of Anr may benefit P. aeruginosa in vivo through the induction of high-affinity terminal oxidases that can increase respiration at low oxygen tensions and may also give P. aeruginosa an advantage, as oxygen becomes limiting during biofilm formation or depletion of local oxygen by the host immune response (55).…”

cbb 3 -Type Cytochrome c Oxidases, Aerobic Respiratory Enzymes, Impact the Anaerobic Life of Pseudomonas aeruginosa PAO1

“…For instance, NO can promote membrane vesicle (MV) production and type III secretion system (T3SS) expression, which are involved in host infection (15,33). NO not only promotes biofilm formation under anoxic conditions (26) but also functions as a signal to induce biofilm dispersal under certain conditions (28).…”

The Moraxella catarrhalis Nitric Oxide Reductase Is Essential for Nitric Oxide Detoxification

“…Recently, denitrification by bacterial pathogens has attracted increasing interest (51). The nitrate reductase complex NarGHJI was shown to be involved in Mycobacterium bovis BCG virulence in an animal model (73) and was implicated in Pseudomonas aeruginosa biofilm formation and virulence (65,66). The Brucella melitensis denitrification pathway is required for virulence in mice (6,23).…”