Nitrite reductase is critical for Pseudomonas aeruginosa survival during co-infection with the oral commensal Streptococcus parasanguinis

Abstract

Pseudomonas aeruginosa is the major aetiological agent of chronic pulmonary infections in cystic fibrosis (CF) patients. However, recent evidence suggests that the polymicrobial community of the CF lung may also harbour oral streptococci, and colonization by these micro-organisms may have a negative impact on P. aeruginosa within the CF lung. Our previous studies demonstrated that nitrite abundance plays an important role in P. aeruginosa survival during co-infection with oral streptococci. Nitrite reductase is a key enzyme involved in nitrite metabolism. Therefore, the objective of this study was to examine the role nitrite reductase (gene nirS) plays in P. aeruginosa survival during co-infection with an oral streptococcus, Streptococcus parasanguinis. Inactivation of nirS in both the chronic CF isolate FRD1 and acute wound isolate PAO1 reduced the survival rate of P. aeruginosa when co-cultured with S. parasanguinis. Growth of both mutants was restored when co-cultured with S. parasanguinis that was defective for H₂O₂ production. Furthermore, the nitrite reductase mutant was unable to kill Drosophila melanogaster during co-infection with S. parasanguinis. Taken together, these results suggest that nitrite reductase plays an important role for survival of P. aeruginosa during co-infection with S. parasanguinis.

Introduction

Commensal and pathogenic bacteria frequently dwell within polymicrobial infections, and engage in interactions that impact both pathogenesis and the host response (Ramsey & Whiteley, 2009). Often, these consortia of bacteria work in concert to promote infection, cope with environmental stress, metabolize nutrients or resist clearing by the host immune response (Peters et al., 2012; Stacy et al., 2014). However, there are instances when bacteria living in these communities are in direct conflict. Within these polymicrobial environments several factors can influence the balance of power between microbes, including bacterial self-defence weapons such as bacteriocins, secondary metabolites or H₂O₂ (Kreth et al., 2008; Liu et al., 2011). Interestingly, host-derived nutrients can also play a role in promoting bacterial competition, as in the case of oral commensal streptococci and Pseudomonas aeruginosa.

Oral commensal streptococci are abundant in the oral cavity, and because they are early colonizers of the tooth surface, they play a role in facilitating the colonization of cariogenic bacteria such as Streptococcus mutans (Kolenbrander & London, 1993; Nobbs et al., 2009). Interestingly, viridans streptococci, including Streptococcus parasanguinis, Streptococcus gordonii and Streptococcus sanguinis, have been detected in the sputum of cystic fibrosis (CF) patients (Maeda et al., 2011). Moreover, some oral commensal bacteria have been associated with improved lung function in patients with CF (Filkins et al., 2012). Historically, P. aeruginosa has been a dominant pathogen during pulmonary CF infections, and has contributed to morbidity and mortality (Smith et al., 2006). P. aeruginosa is a major pathogen of multiple hosts, causes both acute and chronic infections, and has also been detected in some unique cases of periodontitis (Majorana et al., 1999). In addition to being multidrug-resistant, P. aeruginosa has the extraordinary ability to persist during an infection by altering the regulation of virulence and metabolic gene expression, with the most notable example being during CF pulmonary infections (Carmeli et al., 1999). The association between oral commensal streptococci in the CF lung and improved lung function suggested that some oral commensal streptococci could potentially interfere with the pathogenesis of P. aeruginosa. Previously, our laboratory reported that H₂O₂-producing oral commensal streptococci can inhibit P. aeruginosa in a nitrite-dependent manner through the production of reactive nitrogenous species (RNS) (Scoffield & Wu, 2015). Nitrite is readily available within the oral cavity and CF lung, and is a byproduct of denitrifying bacteria (Grasemann et al., 1998; Hezel & Weitzberg, 2015). Nitrite reductase is one of several enzymes unique to the denitrification pathway of P. aeruginosa and is responsible for catalysing the reduction of nitrite to nitric oxide. As nitrite is crucial for the killing of P. aeruginosa by oral commensal streptococci, we questioned the role that the nitrite reductase of P. aeruginosa plays in facilitating its susceptibility to an oral commensal streptococcus, S. parasanguinis.

We report that P. aeruginosa isolates that are defective in nitrite reductase are susceptible to killing by S. parasanguinis. In addition, the inhibition of P. aeruginosa nirS mutants was mediated by an accumulation of nitrite in these mutants and by the production of H₂O₂ in S. parasanguinis. In addition, loss of nirS decreased the fitness of P. aeruginosa in a Drosophila melanogaster infection model and co-infection with S. parasanguinis further attenuated P. aeruginosa virulence in this model. In summary, our study demonstrates the importance of nirS for P. aeruginosa pathogenesis and survival during polymicrobial infections with S. parasanguinis. Furthermore, it highlights the importance of understanding how polymicrobial interactions play a critical role in shaping the outcomes of disease.

Methods

Bacterial strains, culture conditions and reagents

Bacterial strains and plasmids are listed in Table 1. P. aeruginosa was isolated on Pseudomonas Isolation Agar (PIA; Difco) and subsequently cultured in Luria broth (L-broth; Fisher) and incubated at 37 °C. Escherichia coli cells were also cultured in L-broth and incubated 37 °C. Oral streptococci were routinely cultured in Todd–Hewitt broth (THB; Difco) and incubated at 37 °C with 5 % CO₂. Antibiotics were purchased from Sigma-Aldrich and used at the following concentrations: 100 μg ampicillin ml^− 1 for E. coli; 125 μg kanamycin ml^− 1 and 10 μg erythromycin ml^− 1 for S. parasanguinis; and 100 μg gentamicin ml^− 1 and 100 μg carbenicillin ml^− 1 for P. aeruginosa. Peroxynitrite was purchased from Cayman Chemical.

Table 1. Bacterial strains and plasmids used in this study.

Strain or plasmid	Relevant characteristics	Reference or source
S. parasanguinis
FW213	S. parasanguinis parent strain	Cole et al. (1976)
FW213 poxL	Pyruvate oxidase/H2O2 mutant	Scoffield & Wu (2015)
FW213 poxL C	Pyruvate oxidase/H2O2 complemented	Scoffield & Wu (2015)
P. aeruginosa
PAO1	Wound isolate, non-mucoid	Holloway et al. (1979)
FRD1	CF isolate, mucoid	Ohman & Chakrabarty (1981)
PAO1 nirS	Nitrite reductase mutant	This study
FRD1 nirS	Nitrite reductase mutant	This study
PAO1 nirS C	Nitrite reductase complemented	This study
FRD1 nirS C	Nitrite reductase complemented	This study
Plasmids
pSS223	Transcriptional fusion	Suh et al. (2004)
pBluescript K(+)	Cloning vector	Addgene

Competition assays on solid medium and in liquid medium

To examine the interactions between the oral streptococcal species and P. aeruginosa, a 10 μl subculture of each streptococcal species was inoculated onto a Todd–Hewitt agar (THA; Difco) plate as the early colonizer. After incubation overnight at 37 °C with 5 % CO₂, 10 μl of subcultured FRD1 (chronic P. aeruginosa CF isolate) or PAO1 (acute P. aeruginosa isolate) was inoculated next to the streptococci as the late colonizer. The plate was incubated overnight at 37 °C with 5 % CO₂. Growth inhibition of P. aeruginosa was assessed by the presence of a proximal zone of inhibition at the intersection with the early colonizer.

For competition assays in liquid medium, all streptococcal species were grown in THB overnight and 5 μl of cells were subcultured in a Costar 96-well microtitre plate (Corning) containing 200 μl fresh THB and grown to OD₄₇₀ 0.1, followed by the addition of 5 μl P. aeruginosa subculture (OD₆₀₀ 0.2–0.3). The cells were incubated overnight at 37 °C in the presence of 5 % CO₂. P. aeruginosa and oral streptococci cells were dispersed by vigorous pipetting, serially diluted and plated on PIA or THA in duplicate, and the c.f.u. counts were determined the next day.

Construction of the P. aeruginosa nirS mutant and complemented strains

To generate mutants of nirS, a DNA sequence containing ∼500 bp upstream and 500 bp downstream of the nirS coding sequence was PCR amplified from FRD1 cells and cloned into the SmaI site of pBluescript K(+). An internal 800 bp fragment of the nirS coding sequence was removed using inverse PCR and replaced with the aacC1 gene encoding gentamicin resistance (Schweizer, 1993) as a SmaI fragment. This was followed by introduction of an origin of transfer (moriT) of RP4 on a 230 bp HindIII fragment. The resulting plasmid was introduced into P. aeruginosa strains PAO1 and FRD1 by triparental mating, and potential nirS mutants were isolated as gentamicin-resistant, carbenicillin-sensitive colonies, indicating a double-crossover event. Replacement of the WT nirS gene with the nirS : : aacC1 allele was verified by PCR analysis. To complement the nirS mutant, the full-length nirS was PCR-purified from FRD1 cells and cloned into the SmaI site of pBluescript K(+). The resulting plasmid was converted to a mobilizable plasmid via the addition of a moriT in the HindIII site and then introduced into P. aeruginosa through triparental mating.

Construction of nirS transcriptional fusions

The nirS : : lacZ transcriptional fusions were constructed using the nirS gene fragments isolated from PAO1 and FRD1. The fragments, which included 500 bp upstream from the coding sequence, were cloned into the SmaI site of pSS223 (Suh et al., 2004). The plasmids containing the 5′ coding sequence for nirS, in the proper orientation, were verified by PCR and restriction digest. The plasmids containing the fusions were conjugated into FRD1 and PAO1 via triparental mating, and plasmid integration events were selected by carbenicillin resistance.

S. parasanguinis and P. aeruginosa oral infection of D. melanogaster

D. melanogaster were maintained on Jazz-mix Drosophila fly food (Fisher). To orally infect Drosophila, P. aeruginosa (PAO1) and S. parasanguinis were each grown to OD₆₀₀ 2.0, and 0.75 ml culture was centrifuged at 5000 g for 10 min to pellet cells. The bacterial pellet was resuspended in 100 μl of sterile 5 % sucrose. The resuspended cells were spotted onto a of sterile 21 mm filter (Whatman) that was placed on the surface of 5 ml solidified 5 % sucrose agar in a plastic vial (FlyBase). The filters were allowed to dry at room temperature for ∼30 min before addition of Drosophila. To ensure maximum feeding on the discs containing bacteria, male Canton S flies (1–3 days old) were starved for 3 h before being added to vials (10 flies per vial). Flies were anaesthetized using CO₂ throughout the sorting and transferring process. The number of live flies to start the experiment was documented and live flies were counted at 24 h intervals.

Biochemical assays

β-Galactosidase assays were performed as described by Miller (1972). Nitrite concentrations were measured using the Griess reagent (Promega).

Qualitative real-time (qRT)-PCR

RNA was extracted from exponential-phase P. aeruginosa cultures using a Direct-zol kit (Zymo Research). Residual DNA was digested using RQ1 DNase (Promega). RNA was purified with an RNeasy Mini kit (Qiagen), and converted into cDNA using an iScript cDNA Synthesis kit (Bio-Rad). cDNA was then used for qRT-PCR with iQ SYBR Green Supermix (Bio-Rad). The primers used to amplify 16S rRNA (reference) and the nirS gene were forward-GCTGGACTATCGCCGCTG/reverse-ATCTCGTAACCGGTGAAGGTG and forward-TGAAGTCTGGTTCTCGGTGTG/reverse-TCGTGCTGGGTGTTGTAGAC, respectively.

Statistical analysis

Statistical significance was determined using Student's t-test or the log-rank test. Data were considered statistically significant if P < 0.05.

Results

P. aeruginosa isolates deficient for nitrite reductase are susceptible to S. parasanguinis-mediated killing

We previously reported that exogenous nitrite facilitates the inhibition of P. aeruginosa by H₂O₂-producing oral streptococci via the generation of a reactive nitrogenous intermediate (Scoffield & Wu, 2015). The denitrification pathway of P. aeruginosa contains a host of enzymes devoted to converting nitrate to nitrous oxide and finally nitrogen. Nitrite reductase is the second enzyme in this pathway and is responsible for the conversion of nitrite to nitric oxide (Fig. 1a). Based on our previous study, we hypothesized that the activity of nitrite reductase could potentially be a major factor that contributed to the streptococcal and nitrite-mediated inhibition of P. aeruginosa due to an overproduction of RNS such as peroxynitrite or nitric oxide; hence, loss of nitrite reductase would result in increased P. aeruginosa survival due to a decreased production of RNS. Therefore, we wanted to explore the role that the nitrite reductase of P. aeruginosa plays during interactions with S. parasanguinis strain FW213. We constructed a mutation in the nirS gene that encodes nitrite reductase in both an acute (PAO1) and chronic (FRD1) isolate of P. aeruginosa. Next, we tested the fitness of this mutant in a plate competition assay with S. parasanguinis. As shown in Fig. 1(b), nirS mutations in both the acute and chronic P. aeruginosa isolates were sensitive to inhibition by S. parasanguinis compared with the WT and complemented strains. We previously reported that pyruvate oxidase, an enzyme required for the production of H₂O₂ in oral commensal streptococci, is required for S. parasanguinis and nitrite-mediated inhibition of P. aeruginosa. Therefore, we tested whether poxL, the gene that encodes pyruvate oxidase, facilitated inhibition of the P. aeruginosa nirS mutant during P. aeruginosa co-culture with S. parasanguinis. Loss of nirS resulted in a 2 and 4 log decrease in P. aeruginosa survival in the PAO1 and FRD1 isolates, respectively, when co-cultured with WT S. parasanguinis (Fig. 1c, d), even without the addition of exogenous nitrite. Growth of the nirS mutants was rescued by nirS complementation or during growth with the S. parasanguinis poxL mutant. Remarkably, co-culture of the P. aeruginosa nirS mutant with the S. parasanguinis poxL complemented strain resulted in complete inhibition of P. aeruginosa survival (Fig. 1c, d). Taken together, these results suggested that loss of nirS in P. aeruginosa did not abolish the production of potential nitrogenous byproducts that might promote inhibition by H₂O₂-producing streptococci.

Fig. 1.

Nitrite reductase is required for P. aeruginosa survival during co-culture with S. parasanguinis strain FW213. (a) Denitrification pathway of P. aeruginosa. (b) Plate competition assay of S. parasanguinis strain FW213 with P. aeruginosa acute isolate PAO1 or P. aeruginosa chronic CF isolate FRD1. (c) Co-culture of FW213 and poxL mutant and complemented strains with PAO1 WT, nirS mutant and complemented strains. (d) Co-culture of FW213 and poxL mutant and complemented strains with FRD1 WT, nirS mutant and complemented strains. Data are means ± sd and are representative of three experiments. *P < 0.05; **P < 0.005 (Student's t-test). nd, Not detected.

Loss of nitrite reductase increases NO₂ levels in P. aeruginosa

Due to the increased sensitivity of the P. aeruginosa nirS mutant to S. parasanguinis without the addition of exogenous nitrite, we questioned whether the loss of nirS resulted in an accumulation of intracellular nitrite, which would explain why P. aeruginosa is sensitive to nitrite and streptococcal-mediated activity, as reported in our previous study (Scoffield & Wu, 2015). We measured intracellular and extracellular nitrite concentrations in mid-exponential-phase cultures of the PAO1 and FRD1 WT and nirS mutants. As shown in Fig. 2, the loss of nirS in PAO1 resulted in a significant increase in extracellular nitrite compared with WT PAO1. Interestingly, loss of nirS in FRD1 resulted in an increase in both intracellular and extracellular nitrite. A build-up of nitrite in the P. aeruginosa nirS mutant would contribute to the generation of reactive nitrogenous intermediates and increase the susceptibility of P. aeruginosa to killing by S. parasanguinis. These data were consistent with our previous study, which demonstrated that exogenous nitrite facilitated the generation of reactive nitrogenous intermediates when P. aeruginosa was co-cultured with H₂O₂-producing oral streptococci. Moreover, we previously observed that the chronic CF isolate FRD1 was more sensitive to oral streptococci and nitrite-mediated activity compared with the acute PAO1, as was the case with the FRD1 nirS mutant. Taken together, these observations implied that different P. aeruginosa isolates may have altered nitrite reductase activity.

Fig. 2.

Loss of nitrite reductase in P. aeruginosa results in an accumulation of NO₂. Data are means ± sd and are representative of three experiments. *P < 0.05 (Student's t-test).

Expression of nirS is reduced in the FRD1 isolate compared with the PAO1 isolate in response to nitrogenous intermediates and exposure to S. parasanguinis and nitrite

Previously, our laboratory reported that the FRD1 isolate displayed increased sensitivity to oral streptococci and nitrite-mediated activity compared with PAO1, which suggested that this isolate may be deficient in eliciting a response to nitrosative stress or was ineffective in reducing nitrite. Therefore, we constructed nirS : : lacZ transcriptional fusions in FRD1 and PAO1 to monitor promoter activity of nirS when grown on L-broth or L-broth that contained 2 mM H₂O₂, 1 mM NO₂, 1 mM NO₃ or 250 μM ONOO⁻ (peroxynitrite). We also measured nirS expression in P. aeruginosa and S. parasanguinis co-cultures ( ± 1 mM NO₂) using qRT-PCR analysis. As shown in Fig. 3(a), nirS expression was not induced in FRD1 compared with PAO1 when exposed to NO₂, NO₃ or ONOO⁻. In addition, nirS expression was not induced by the presence of H₂O₂ in either isolate. Furthermore, qRT-PCR analysis demonstrated that nirS expression was induced in PAO1, but not FRD1, when P. aeruginosa was co-cultured with S. parasanguinis in the presence of nitrite (Fig. 3b). It is important to note that catalase (katA) has been shown to be involved in P. aeruginosa resistance to nitric oxide (Su et al., 2014); however, we measured katA expression in WT PAO1 and FRD1 and in the nirS mutants, and although the nirS mutants were marginally reduced in katA expression, we found no difference in katA expression when the WT isolates and nirS mutants were co-cultured with S. parasanguinis (data not shown). Altogether, these data suggested that nirS was largely induced by nitrogenous intermediates and not H₂O₂ produced by S. parasanguinis, and that nirS played a role in alleviating endogenous nitrogenous stress. Reduced activity of nitrite reductase by FRD1 during co-culture with S. parasanguinis and nitrite might have contributed to this isolate's increased sensitivity to S. parasanguinis nitrite-mediated activity as previously reported, and signified the importance of this enzyme for the survival of P. aeruginosa during polymicrobial infections with oral streptococci.

Fig. 3.

Nitrite reductase expression is increased in response to nitrogenous intermediates in PAO1, but not FRD1. (a) nirS expression was measured in cultures of P. aeruginosa grown in L-broth that contained 2 mM H₂O₂, 1 mM NO₂, 1 mM NO₃ or 250 μM ONOO⁻ (peroxynitrite). (b) nirS expression was measured in P. aeruginosa and FW213 co-cultures ( ± 1 mM nitrite). Data are means ± sd and are representative of three experiments.

Loss of nirS in P. aeruginosa results in increased D. melanogaster survival during co-infection with S. parasanguinis

P. aeruginosa isolates defective for nirS display reduced survival in the presence of S. parasanguinis; thus, we wanted to test whether a mutation in nirS would render P. aeruginosa less fit in the Drosophila in vivo model of infection during co-infection with S. parasanguinis. P. aeruginosa strain PAO1, but not S. parasanguinis, readily kills Drosophila, and we previously established a Drosophila co-infection model to study P. aeruginosa and S. parasanguinis in vivo interactions (Scoffield & Wu, 2015). We infected Drosophila with WT PAO1 and the PAO1 nirS mutant, and also co-infected Drosophila with PAO1 and the PAO1 nirS mutant that contained equivalent numbers of S. parasanguinis. Compared with PAO1, the PAO1 nirS mutant displayed reduced virulence in the infection model. Following 8 days of infection, the survival rate for the PAO1 nirS mutant was 70 %, compared with 0 % in PAO1. However, when Drosophila were co-infected with the PAO1 nirS mutant and S. parasanguinis, the survival rate increased to 100 % (Fig. 4a). Enumeration of P. aeruginosa in Drosophila revealed that PAO1 was able to better colonize the flies compared with the PAO1 nirS mutant. When we compared the number of P. aeruginosa bacteria remaining in the flies following co-infection, the PAO1 nirS mutant that was co-infected with S. parasanguinis had a severe reduction in c.f.u. compared with the PAO1 nirS mutant bacteria used for the single infection. Lastly, co-infection with S. parasanguinis did not affect the colonization of WT PAO1 in Drosophila (Fig. 4b). These data suggested that a functional nirS was critical for P. aeruginosa pathogenesis and was also important during polymicrobial infections with S. parasanguinis.

Fig. 4.

Nitrite reductase is required for the killing of D. melanogaster by PAO1 in the presence of FW213. (a) Drosophila were infected with WT PAO1 or the PAO1 nirS mutant, or co-infected with S. parasanguinis FW213. (b) P. aeruginosa c.f.u. counts during the single or co-infection. Data are mean ± sd and are representative of four biological replicates. n = 40. *P < 0.05, **P < 0.005 (log-rank test).

Discussion

Oral commensal streptococci, including S. parasanguinis, have been shown to be prevalent in CF pulmonary infections (Maeda et al., 2011), which historically have been dominated by the presence of the major CF pathogen P. aeruginosa. Interestingly, the occurrence of oral commensal streptococci in the CF lung environment has been associated with improved lung function (Filkins et al., 2012); however, the mechanism by which S. parasanguinis benefits CF patients is unclear. We previously reported that exogenous nitrite facilitates the inhibition of P. aeruginosa by S. parasanguinis due to the production of reactive nitrogenous intermediates and the inhibition of P. aeruginosa in this manner could be beneficial for the host (Scoffield & Wu, 2015). In this study, we hypothesized that nitrite reductase, a P. aeruginosa enzyme involved in denitrification, could potentially be involved in the resistance or susceptibility of P. aeruginosa to reactive nitrogenous intermediates generated by S. parasanguinis activity. Therefore, we examined the role nitrite reductase plays in P. aeruginosa survival during co-infection with S. parasanguinis. Here, we report that loss of nitrite reductase promotes the accumulation of nitrite in P. aeruginosa cultures and thus renders P. aeruginosa nirS mutants sensitive to killing by H₂O₂-producing S. parasanguinis. These data are consistent with our previous report in which exogenous nitrite promoted the inhibition of P. aeruginosa by S. parasanguinis in an H₂O₂-dependent manner. Furthermore, the P. aeruginosa nirS mutant is less fit in the Drosophila melanogaster in vivo model of infection and, in addition, co-infection with S. parasanguinis in Drosophila further decreased the fitness of the nirS mutant. Taken together, our results demonstrate that nitrite reductase is critical for P. aeruginosa survival during polymicrobial infections with the oral commensal streptococcus S. parasanguinis and colonization of the host.

Nitrogenous intermediates generated within infection sites are often host-derived or are the result of nitrite or nitrate metabolism by denitrifying bacteria. Although little is known about how nitrogenous intermediates or denitrifying mechanisms influence competition between diverse micro-organisms within a polymicrobial environment, our study suggests denitrification processes may promote bacterial competition and could potentially impact disease outcomes in a manner that may be beneficial to the host. Nitrite is readily available in the human body for conversion to nitrogenous intermediates and has been linked to improved health, particularly in the oral cavity (Hyde et al., 2014). Elevated salivary nitrite concentrations have been associated with a reduction in dental caries, presumably due to the inhibition of the cariogenic pathogen S. mutans by nitric oxide-generating oral commensal bacteria (Doel et al., 2004). It is hypothesized that nitric oxide (or other RNS) generated by denitrifying oral commensals may modulate microbial homeostasis (Hyde et al., 2014), and thereby function as an infection control strategy. However, it is important to note that polymicrobial interactions involving P. aeruginosa and oral streptococci in the CF lung are complex, and are often controlled by the dynamics of specific streptococcal populations, colonization sequence and environmental conditions. For example, streptococcal species that belong to the Streptococcus milleri and Streptococcus anginosus groups have also been found to be co-colonized with P. aeruginosa in the CF airway and upregulate P. aeruginosa virulence, and as a result are considered pathogens in some cases of CF (Parkins et al., 2008; Sibley et al., 2008; Whiley et al., 2014). Furthermore, colonization sequence is also important for the ability of oral commensal streptococci to inhibit P. aeruginosa. S. gordonii and S. sanguinis inhibit the P. aeruginosa Liverpool epidemic strain in an H₂O₂-dependent manner when these streptococci are inoculated as the primary colonizer in the presence of CO₂ (Whiley et al., 2015), which is consistent with our previous and current studies that demonstrate H₂O₂-producing streptococci inhibit a chronic and acute isolate of P. aeruginosa in the presence of nitrite when oral commensal streptococci are the primary colonizers (Scoffield & Wu, 2015). Overall, more studies are required to strengthen our understanding of how H₂O₂-producing commensal streptococci and nitrite can negatively impact P. aeruginosa virulence.

Micro-organisms are continuously exposed to nitrosative stress and are equipped with mechanisms that function to alleviate this stress encountered within the host. Moreover, the altered or reduced expression of denitrifying enzymes, as reported in this study using the P. aeruginosa FRD1 clinical isolate, could render some bacterial isolates more susceptible to RNS stress mediated by the host or other bacteria. Functional denitrification enzymes have been previously reported to be crucial for the survival of other bacterial pathogens. For example, Mycobacterium tuberculosis survival in human macrophages is dependent upon a functional nitrite reductase (NirBD) (Akhtar et al., 2013). Moreover, HcpR, a regulator of hydroxylamine reductase, is required for Porphyromonas gingivalis growth in the presence of nitrite and nitric oxide, in addition to survival in host cells (Lewis et al., 2012). Furthermore, nitrite reductase has been shown to be required for the expression of the P. aeruginosa type III secretion system (Van Alst et al., 2009) and biofilm formation by P. aeruginosa (de la Fuente-Núñez et al., 2013). These studies suggest that mechanisms involved in nitrosative stress resistance or nitrite reduction are required for not only bacterial growth on nitrogenous substrates, but also bacterial virulence.

Few studies have examined the role that denitrification mechanisms have on bacterial competition within polymicrobial infections. Our findings demonstrate that nitrite reductase is required by P. aeruginosa in order to compete with the oral commensal S. parasanguinis. In addition, oral commensal bacteria may mediate antimicrobial mechanisms not only in the oral cavity, but also during systemic infections such as CF where P. aeruginosa and oral commensal streptococci are co-colonized. Furthermore, any alterations in the expression of enzymes that are important for the resistance or detoxification of nitrogenous intermediate could render micro-organisms susceptible to killing within a polymicrobial infection. Understanding the mechanisms that are required for pathogens to persist during polymicrobial infections could potentially lead to the development of novel therapeutics that promote bacterial competition and result in positive disease outcomes.

Abbreviations:

CF

cystic fibrosis

qRT

quantitative real-time

RNS

reactive nitrogenous species.