The putative bacterial oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) suppresses antibiotic resistance and pathogenicity in Pseudomonas aeruginosa

Abstract

Pseudomonas aeruginosa is an extracellular opportunistic bacterial pathogen commonly associated with infectious complications in susceptible individuals, such as those with underlying diseases including HIV/AIDS and cystic fibrosis. Antibiotic resistance in multiple strains of P. aeruginosa is a rapidly developing clinical problem. We have previously demonstrated that the oxygen levels at the site of P. aeruginosa infection can strongly influence virulence and antibiotic resistance in this pathogen, although the oxygen-sensing and -signaling mechanisms underpinning this response have remained unknown. In this study, we investigated the potential role of the putative oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) in the control of virulence and antibiotic resistance in P. aeruginosa. We found that a P. aeruginosa strain lacking PPHD (PAO310) exhibits increased virulence associated with increased bacterial motility. Furthermore, PPHD-deficient P. aeruginosa displayed enhanced antibiotic resistance against tetracycline through increased expression of the xenobiotic transporters mexEF-oprN and MexXY. Of note, the effect of the PPHD knockout on antibiotic resistance was phenocopied in bacteria exposed to atmospheric hypoxia. We conclude that PPHD is a putative bacterial oxygen sensor that may link microenvironmental oxygen levels to virulence and antibiotic resistance in P. aeruginosa. Pseudomonas aeruginosa is an extracellular opportunistic bacterial pathogen commonly associated with infectious complications in susceptible individuals, such as those with underlying diseases including HIV/AIDS and cystic fibrosis. Antibiotic resistance in multiple strains of P. aeruginosa is a rapidly developing clinical problem. We have previously demonstrated that the oxygen levels at the site of P. aeruginosa infection can strongly influence virulence and antibiotic resistance in this pathogen, although the oxygen-sensing and -signaling mechanisms underpinning this response have remained unknown. In this study, we investigated the potential role of the putative oxygen sensor Pseudomonas prolyl hydroxylase (PPHD) in the control of virulence and antibiotic resistance in P. aeruginosa. We found that a P. aeruginosa strain lacking PPHD (PAO310) exhibits increased virulence associated with increased bacterial motility. Furthermore, PPHD-deficient P. aeruginosa displayed enhanced antibiotic resistance against tetracycline through increased expression of the xenobiotic transporters mexEF-oprN and MexXY. Of note, the effect of the PPHD knockout on antibiotic resistance was phenocopied in bacteria exposed to atmospheric hypoxia. We conclude that PPHD is a putative bacterial oxygen sensor that may link microenvironmental oxygen levels to virulence and antibiotic resistance in P. aeruginosa. Humans exist in a state of communion with commensal microbial organisms including thousands of species of bacteria that provide essential support for the growth and development of organs including the skin and intestine and the maturation of the host immune system. These bacteria comprise the human microbiome. In the vast majority of cases, interactions between humans and bacteria result in a neutral or beneficial outcome for the host. However, in a limited number of cases, microbes display the capacity for virulence and pathogenicity, and this can lead to tissue damage in the host, resulting in infectious disease (1Cohen J. Powderly W. Infectious Diseases. Mosby Press, St. Louis, MO2010Google Scholar). Our capacity to clinically manage opportunistic bacterial infection is rapidly diminishing because many of the causative pathogens develop antibiotic resistance. Pseudomonas aeruginosa, a motile, Gram-negative facultative anaerobe, is an opportunistic pathogen that is responsible for 17% of cases of nosocomial pneumonia and is a primary cause of morbidity and mortality in cystic fibrosis patients (2Fujitani, S., Moffett, K. S., and Yu, V. L., (n.d.) Pseudomonas aeruginosa, . Antimicrobe, Pittsburgh, PA, 15219.Google Scholar, 3Locke L.W. Myerburg M.M. Weiner D.J. Markovetz M.R. Parker R.S. Muthukrishnan A. Weber L. Czachowski M.R. Lacy R.T. Pilewski J.M. Corcoran T.E. Pseudomonas infection and mucociliary and absorptive clearance in the cystic fibrosis lung.Eur. Respir. J. 2016; 47 (27009167): 1392-140110.1183/13993003.01880-2015Crossref PubMed Scopus (17) Google Scholar). P. aeruginosa infection is also frequently associated with infections in individuals with HIV/AIDS, urinary tract infections, ventilator-associated lung infection, endocarditis, meningitis, ocular infection, ear infection, and wound/burn/skin infections (2Fujitani, S., Moffett, K. S., and Yu, V. L., (n.d.) Pseudomonas aeruginosa, . Antimicrobe, Pittsburgh, PA, 15219.Google Scholar). P. aeruginosa is an extracellular pathogen that produces toxins (e.g. exotoxin A, which causes direct tissue damage) and proteases (e.g. alkaline protease) that degrade neutrophil-derived elastin and consequently help the bacterium to avoid host immunity. P. aeruginosa expresses a type III secretion system that allows the direct injection of bacterial toxins into host cells, siderophores that promote the chelation of iron and virulence factors such as pyocyanin and pyoverdine, which support bacterial metabolism and the production of exotoxins, respectively. Together, these virulence mechanisms promote bacterial colonization of tissues and the development of infectious disease. A key mechanism underpinning the development of virulence in P. aeruginosa is quorum-sensing, which refers to the intercellular communication between individual bacteria in the niche of the infected tissue (4Daniels R. Vanderleyden J. Michiels J. Quorum sensing and swarming migration in bacteria.FEMS Microbiol. Rev. 2004; 28 (15449604): 261-28910.1016/j.femsre.2003.09.004Crossref PubMed Scopus (435) Google Scholar). Several important factors that mediate the dialog between bacteria during quorum sensing are the products of bacterial metabolism creating another link between the bacterial metabolic strategy and the capacity of the pathogen for virulence. Antimicrobial resistance is a common and developing problem in P. aeruginosa infection caused in part by high expression levels of a number of xenobiotic efflux pumps (5Schaffer K. Taylor C.T. The impact of hypoxia on bacterial infection.FEBS J. 2015; 282 (25786849): 2260-226610.1111/febs.13270Crossref PubMed Scopus (77) Google Scholar, 6Schaible B. Taylor C.T. Schaffer K. Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.Antimicrob. Agents Chemother. 2012; 56 (22290986): 2114-211810.1128/AAC.05574-11Crossref PubMed Scopus (74) Google Scholar). Typically, when it colonizes a host, P. aeruginosa grows in a biofilm that provides a physical barrier to antimicrobial access. In summary, P. aeruginosa is a formidable opportunistic pathogen that is armed with an array of features that support its virulence in human infection. Hypoxia is an environmental feature that is frequently encountered at sites of infection (5Schaffer K. Taylor C.T. The impact of hypoxia on bacterial infection.FEBS J. 2015; 282 (25786849): 2260-226610.1111/febs.13270Crossref PubMed Scopus (77) Google Scholar). For example, the cystic fibrosis lung, commonly infected with P. aeruginosa, has been shown to be hypoxic (7Worlitzsch D. Tarran R. Ulrich M. Schwab U. Cekici A. Meyer K.C. Birrer P. Bellon G. Berger J. Weiss T. Botzenhart K. Yankaskas J.R. Randell S. Boucher R.C. Döring G. Effects of reduced mucus oxygen concentration in airway Pseudomonas infections of cystic fibrosis patients.J. Clin. Invest. 2002; 109 (11827991): 317-32510.1172/JCI0213870Crossref PubMed Scopus (979) Google Scholar). Furthermore, Mycobacterium tuberculosis was shown to reside in a hypoxic environment within tuberculous granulomas in rabbits (8Via L.E. Lin P.L. Ray S.M. Carrillo J. Allen S.S. Eum S.Y. Taylor K. Klein E. Manjunatha U. Gonzales J. Lee E.G. Park S.K. Raleigh J.A. Cho S.N. McMurray D.N. et al.Tuberculous granulomas are hypoxic in guinea pigs, rabbits, and nonhuman primates.Infect. Immun. 2008; 76 (18347040): 2333-234010.1128/IAI.01515-07Crossref PubMed Scopus (494) Google Scholar). Biopsies from patients with skin infection showed an elevated level of hypoxia-inducible factor (HIF) 1α, a marker of tissue hypoxia (9Werth N. Beerlage C. Rosenberger C. Yazdi A.S. Edelmann M. Amr A. Bernhardt W. von Eiff C. Becker K. Schäfer A. Peschel A. Kempf V.A. Activation of hypoxia inducible factor 1 is a general phenomenon in infections with human pathogens.PLoS One. 2010; 5 (20644645): e1157610.1371/journal.pone.0011576Crossref PubMed Scopus (130) Google Scholar). We previously demonstrated the impact of hypoxia on P. aeruginosa antibiotic resistance, virulence, and infection (6Schaible B. Taylor C.T. Schaffer K. Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.Antimicrob. Agents Chemother. 2012; 56 (22290986): 2114-211810.1128/AAC.05574-11Crossref PubMed Scopus (74) Google Scholar, 10Schaible B. McClean S. Selfridge A. Broquet A. Asehnoune K. Taylor C.T. Schaffer K. Hypoxia modulates infection of epithelial cells by Pseudomonas aeruginosa.PLoS One. 2013; 8 (23418576): e5649110.1371/journal.pone.0056491Crossref PubMed Scopus (47) Google Scholar, 11Schaible B. Rodriguez J. Garcia A. von Kriegsheim A. McClean S. Hickey C. Keogh C.E. Brown E. Schaffer K. Broquet A. Taylor C.T. Hypoxia reduces the pathogenicity of Pseudomonas aeruginosa by decreasing the expression of multiple virulence factors.J. Infect. Dis. 2017; 215 (28368464): 1459-146710.1093/infdis/jix139Crossref PubMed Scopus (18) Google Scholar). However, the nature of the oxygen-sensing pathways that determine these responses remains unknown and is the topic of the current study. Prolyl hydroxylases (PHDs) 2The abbreviations used are: PHDprolyl hydroxylasePPHDPseudomonas prolyl hydroxylaseMICminimal inhibitory concentrationHIFhypoxia-inducible factorRNDresistance-nodulation-division. are oxygen sensors in metazoans. They are Fe(II)- and 2-oxoglutarate–dependent dioxygenases whose activity depends on molecular oxygen. When sufficient oxygen is available (normoxia), PHDs hydroxylate the hypoxia-inducible factor (HIF) α subunit. The von Hippel–Lindau tumor suppressor protein polyubiquitinates the hydroxylated HIFα and thus marks it for proteosomal degradation. Conversely, when oxygen is limited, HIFα accumulates and translocates to the nucleus where it transcriptionally activates genes involved in erythropoiesis and angiogenesis (12Kaelin Jr., W.G. Ratcliffe P.J. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.Mol. Cell. 2008; 30 (18498744): 393-40210.1016/j.molcel.2008.04.009Abstract Full Text Full Text PDF PubMed Scopus (2192) Google Scholar). prolyl hydroxylase Pseudomonas prolyl hydroxylase minimal inhibitory concentration hypoxia-inducible factor resistance-nodulation-division. PHDs are therefore primary oxygen sensors in metazoans (13Scotti J.S. Leung I.K. Ge W. Bentley M.A. Paps J. Kramer H.B. Lee J. Aik W. Choi H. Paulsen S.M. Bowman L.A. Loik N.D. Horita S. Ho C.H. Kershaw N.J. et al.Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (25197067): 13331-1333610.1073/pnas.1409916111Crossref PubMed Scopus (49) Google Scholar, 14Taylor C.T. McElwain J.C. Ancient atmospheres and the evolution of oxygen sensing via the hypoxia-inducible factor in metazoans.Physiology. 2010; 25 (20940432): 272-27910.1152/physiol.00029.2010Crossref PubMed Scopus (103) Google Scholar). Recently a homolog to the human oxygen sensing enzyme PHD2 was identified in P. aeruginosa and termed Pseudomonas prolyl hydroxylase (PPHD) (13Scotti J.S. Leung I.K. Ge W. Bentley M.A. Paps J. Kramer H.B. Lee J. Aik W. Choi H. Paulsen S.M. Bowman L.A. Loik N.D. Horita S. Ho C.H. Kershaw N.J. et al.Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (25197067): 13331-1333610.1073/pnas.1409916111Crossref PubMed Scopus (49) Google Scholar). Subsequently, another bacterial PHD enzyme was discovered in Bacillus anthracis (15Schnicker N.J. Razzaghi M. Guha Thakurta S. Chakravarthy S. Dey M. Bacillus anthracis prolyl 4-hydroxylase interacts with and modifies elongation factor Tu.Biochemistry. 2017; 56 (28981257): 5771-578510.1021/acs.biochem.7b00601Crossref PubMed Scopus (7) Google Scholar). PPHD has a close structural homology to human PHD2 in that it contains an active 2OG oxygenase domain as demonstrated by biochemical and structural analysis (13Scotti J.S. Leung I.K. Ge W. Bentley M.A. Paps J. Kramer H.B. Lee J. Aik W. Choi H. Paulsen S.M. Bowman L.A. Loik N.D. Horita S. Ho C.H. Kershaw N.J. et al.Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (25197067): 13331-1333610.1073/pnas.1409916111Crossref PubMed Scopus (49) Google Scholar). Furthermore, the elongation factor EF-Tu has been identified as a substrate for PPHD. However, cross-reactivity with substrates does not occur (i.e. EF-Tu is not a substrate for PHD2, and the oxygen-dependent degradation domain of HIF-1α is not a substrate for PPHD). Because of the role of PHDs in mammalian oxygen sensing, it was hypothesized that PPHD might play a role in oxygen sensing in P. aeruginosa (13Scotti J.S. Leung I.K. Ge W. Bentley M.A. Paps J. Kramer H.B. Lee J. Aik W. Choi H. Paulsen S.M. Bowman L.A. Loik N.D. Horita S. Ho C.H. Kershaw N.J. et al.Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (25197067): 13331-1333610.1073/pnas.1409916111Crossref PubMed Scopus (49) Google Scholar, 16Dickinson R.S. Murphy F. Doherty C. Williams S. Mirchandani A. Willson J. Scotti J.S. Preston G. Schofield C.J. Whyte M.K.B. Walmsley S.R. Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitroin vivo.Wellcome Open Res. 2017; 2 (29387803): 10410.12688/wellcomeopenres.12871.1Crossref PubMed Scopus (6) Google Scholar). More recently we and others have shown that PPHD influences P. aeruginosa virulence; however, the mechanisms involved remain to be defined (11Schaible B. Rodriguez J. Garcia A. von Kriegsheim A. McClean S. Hickey C. Keogh C.E. Brown E. Schaffer K. Broquet A. Taylor C.T. Hypoxia reduces the pathogenicity of Pseudomonas aeruginosa by decreasing the expression of multiple virulence factors.J. Infect. Dis. 2017; 215 (28368464): 1459-146710.1093/infdis/jix139Crossref PubMed Scopus (18) Google Scholar, 16Dickinson R.S. Murphy F. Doherty C. Williams S. Mirchandani A. Willson J. Scotti J.S. Preston G. Schofield C.J. Whyte M.K.B. Walmsley S.R. Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitroin vivo.Wellcome Open Res. 2017; 2 (29387803): 10410.12688/wellcomeopenres.12871.1Crossref PubMed Scopus (6) Google Scholar). In this study, we investigated the role of PPHD as a possible prokaryotic oxygen sensor linking microenvironmental hypoxia to virulence and antibiotic resistance in P. aeruginosa. We have previously demonstrated that microenvironmental hypoxia alters virulence in P. aeruginosa (10Schaible B. McClean S. Selfridge A. Broquet A. Asehnoune K. Taylor C.T. Schaffer K. Hypoxia modulates infection of epithelial cells by Pseudomonas aeruginosa.PLoS One. 2013; 8 (23418576): e5649110.1371/journal.pone.0056491Crossref PubMed Scopus (47) Google Scholar, 11Schaible B. Rodriguez J. Garcia A. von Kriegsheim A. McClean S. Hickey C. Keogh C.E. Brown E. Schaffer K. Broquet A. Taylor C.T. Hypoxia reduces the pathogenicity of Pseudomonas aeruginosa by decreasing the expression of multiple virulence factors.J. Infect. Dis. 2017; 215 (28368464): 1459-146710.1093/infdis/jix139Crossref PubMed Scopus (18) Google Scholar). To determine whether the putative oxygen sensor PPHD may play a role in controlling virulence and antibiotic resistance, we compared WT (PAO1) with a P. aeruginosa strain deficient in PPHD (PAO310). We first confirmed the absence of PPHD mRNA expression in PAO310 by PCR (Fig. 1A). We next investigated the impact of PPHD knockout on bacterial virulence using Galleria mellonella larvae as a model of host infection (17Cullen L. Weiser R. Olszak T. Maldonado R.F. Moreira A.S. Slachmuylders L. Brackman G. Paunova-Krasteva T.S. Zarnowiec P. Czerwonka G. Reilly J. Drevinek P. Kaca W. Melter O. De Soyza A. et al.Phenotypic characterization of an international Pseudomonas aeruginosa reference panel: strains of cystic fibrosis (CF) origin show less in vivo virulence than non-CF strains.Microbiology. 2015; 161 (26253522): 1961-197710.1099/mic.0.000155Crossref PubMed Scopus (57) Google Scholar). PAO310 was more virulent in G. mellonella than PAO1 as demonstrated both qualitatively and quantitatively (Fig. 1, B and C, respectively and Fig. S2). A representative image of G. mellonella injected with P. aeruginosa at 24 h is shown in which lighter-colored larvae are viable and darker-colored larvae were melanized, which immediately precedes death. The data in Fig. 1C are presented as LD50 (the number of bacteria required to kill 50% of the larvae). In summary, PPHD knockout results in increased lethality in G. mellonella, indicating that PPHD suppresses bacterial virulence in P. aeruginosa. We next investigated the influence of PPHD expression on key determinants of virulence in P. aeruginosa. We first compared bacterial motility and quantified biofilm formation in PAO1 and PAO310 strains. Consistent with the increased virulence observed in Fig. 1, motility of P. aeruginosa (both swimming (Fig. 2A) and swarming (Fig. 2B)) was increased when PPHD is not present. Furthermore, biofilm mass was also increased in PAO310 (Fig. 2C). Taken together, these data support a role for PPHD in suppressing key determinants of P. aeruginosa virulence. We previously demonstrated that microenvironmental hypoxia plays a determining role in antibiotic resistance in P. aeruginosa (6Schaible B. Taylor C.T. Schaffer K. Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.Antimicrob. Agents Chemother. 2012; 56 (22290986): 2114-211810.1128/AAC.05574-11Crossref PubMed Scopus (74) Google Scholar). Therefore, we next investigated the impact of PPHD knockout on antibiotic resistance. Minimal inhibitory concentration of antibiotics from a broad range of antibiotic classes were tested in PAO1 and PAO310 strains of P. aeruginosa. The minimal inhibitory concentration (MIC) values for tetracycline antibiotics (tetracycline, doxycycline, minocycline, and tigecycline) were selectively increased in P. aeruginosa lacking the PPHD gene, whereas susceptibility of other classes of antibiotics was not influenced (Table 1).Table 1PPHD selectively regulates susceptibility to tetracycline antibioticsAntibiotic classAntibioticPAO1 WT MICPAO310 PPHD KO MICMethodNo. of independent experimentsμg/mlμg/mlβ-LactamsPiperacillin/tazobactam88VITEK2n = 3Aztreonam44E-testn = 1AminoglycosidesAmikacin≤2≤2VITEK2n = 3Gentamycin≤1≤1VITEK2n = 3Tobramycin≤1≤1VITEK2n = 3CarbapenemsMeropenem22VITEK2n = 3Imipenem88VITEK2n = 3Ertapenem≥8≥8MBD (GNX2F)n = 3Doripenem11MBD (GNX2F)n = 3CephalosporinsCefotaxime1616MBD (GNX2F)n = 3Cefepime22VITEK2n = 3Ceftazidime22VITEK2n = 3QuinolonesCiprofloxacin≤0.25≤0.25VITEK2n = 3Levofloxacin0.50.5VITEK2n = 3PenicillinsTicarcillin3232VITEK2n = 3Ticarcillin/clavulanic acid1616VITEK2n = 3PolymyxinsColistin12MBD (GNX2F)n = 3Polymixin B12MBD (GNX2F)n = 3TetracyclinesTetracycline32256MBDn = 4ap < 0.05 by Mann–Whitney test.Doxycycline1664MBDn = 3Minocycline1632MBDn = 3Tigecycline (glycylcycline)816MBDn = 3OthersTrimethoprim/sulfamethoxazole4≥8MBD (GNX2F)n = 3a p < 0.05 by Mann–Whitney test. Open table in a new tab Exposing PAO1 to tetracycline (512 μg/ml) prior to injection into larvae increased its LD50 by 2972-fold (Fig. 3). In contrast, for PAO310, tetracycline pretreatment did not affect its LD50, further suggesting that the P. aeruginosa lacking PPHD was more resistant to the antibiotic. Taken together, these data indicate that PPHD selectively suppresses tetracycline antibiotic resistance in P. aeruginosa. We next investigated the possible mechanisms underpinning the effects of PPHD knockout on antibiotic resistance. A key determinant of antibiotic resistance in P. aeruginosa is the level of antibiotic efflux via RND efflux pumps (18Li X.Z. Livermore D.M. Nikaido H. Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: resistance to tetracycline, chloramphenicol, and norfloxacin.Antimicrob. Agents Chemother. 1994; 38 (7986003): 1732-174110.1128/AAC.38.8.1732Crossref PubMed Scopus (304) Google Scholar, 19Poole K. Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms.J. Mol. Microbiol. Biotechnol. 2001; 3 (11321581): 255-264PubMed Google Scholar). Indeed, we previously demonstrated that the expression of these pumps is increased in hypoxia, thereby contributing to hypoxia-induced antibiotic resistance (6Schaible B. Taylor C.T. Schaffer K. Hypoxia increases antibiotic resistance in Pseudomonas aeruginosa through altering the composition of multidrug efflux pumps.Antimicrob. Agents Chemother. 2012; 56 (22290986): 2114-211810.1128/AAC.05574-11Crossref PubMed Scopus (74) Google Scholar). We next investigated whether RND efflux pumps contribute to increased tetracycline resistance in PAO310. To do this we determined the MIC of tetracycline in the presence of an efflux pump inhibitor (Phe–Arg–β-naphtylamide dihydrochloride). Blocking the RND efflux pumps made PAO310 more susceptible to tetracycline (MIC was lowered from 256 to 64 μg/ml), indicating that the absence of PPHD contributes to antibiotic resistance by increasing tetracycline extrusion via efflux pumps (Fig. 4A). We quantified the expression of the linker protein gene of four RND efflux pumps. MexE (part of mexEF–OprN) was increased in PAO310 (PPHD knockout mutant), whereas mexA and mexC were unchanged (Fig. 4B and Fig. S1). Of note, the induction of mexX with a subinhibitory concentration of tetracycline in PAO1 (WT P. aeruginosa) was lost in PAO310 (PPHD knockout strain) (Fig. S1). These data suggest that the absence of PPHD increases mexEF–OprN expression in P. aeruginosa. PPHD is a prolyl hydroxylase, homolog to human PHD2. PHD2 hydroxylates the target HIF-1α protein in metazoan cells in an oxygen-dependent manner. The hydroxylation activity of PHD2 can therefore be inhibited by low oxygen concentrations (hypoxia) (20Haase V.H. HIF-prolyl hydroxylases as therapeutic targets in erythropoiesis and iron metabolism.Hemodial. Int. 2017; 21 (28449418): S110-S12410.1111/hdi.12567Crossref PubMed Scopus (98) Google Scholar). To investigate whether hypoxia mimics the effects of PPHD knockout on tetracycline antibiotic susceptibility, we tested whether exposure to hypoxia (1% oxygen) altered antibiotic resistance in a similar way to PPHD knockout. Similar to PPHD knockout (albeit to a lesser degree), hypoxia increased tetracycline, doxycycline, and minocycline MICs in PAO1 by ∼2-fold (tetracycline, 32–64 μg/ml; doxycycline and minocycline, 16–32 μg/ml), respectively (Fig. 5A). To test the potential clinical relevance of our observations, we also investigated tetracycline susceptibility in clinical P. aeruginosa isolates (derived from patient infections) under hypoxic conditions. Consistent with the effects observed in PAO1, two of three acute clinical strains demonstrated a 2-fold increase in MIC in hypoxia (Fig. 5B). In summary, exposure to hypoxia phenocopies the impact of PPHD knockout on tetracycline resistance in P. aeruginosa, thereby supporting the potential role for PPHD as a bacterial oxygen sensor. Hypoxia is frequently a prominent microenvironmental feature at sites of infection. We have previously shown that exposure of the opportunistic pathogen P. aeruginosa to a hypoxic microenvironment is a key determinant of pathogen virulence and antibiotic resistance. The mechanism whereby hypoxia mediates its influence on these parameters of pathogen behavior is the topic of this study. Developing our understanding of how the microenvironment at the site of infection affects pathogen virulence and antibiotic resistance will help identify new avenues of therapeutic intervention in P. aeruginosa infection. Recently the prolyl hydroxylase PPHD was identified in P. aeruginosa and hypothesized to play a role in oxygen sensing (13Scotti J.S. Leung I.K. Ge W. Bentley M.A. Paps J. Kramer H.B. Lee J. Aik W. Choi H. Paulsen S.M. Bowman L.A. Loik N.D. Horita S. Ho C.H. Kershaw N.J. et al.Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (25197067): 13331-1333610.1073/pnas.1409916111Crossref PubMed Scopus (49) Google Scholar). PPHD is a structural homolog of human PHD2, which is an important regulator of the hypoxic response in mammals (12Kaelin Jr., W.G. Ratcliffe P.J. Oxygen sensing by metazoans: the central role of the HIF hydroxylase pathway.Mol. Cell. 2008; 30 (18498744): 393-40210.1016/j.molcel.2008.04.009Abstract Full Text Full Text PDF PubMed Scopus (2192) Google Scholar). PPHD was shown to hydroxylate the prokaryotic elongation factor EF-Tu on a proline residue (13Scotti J.S. Leung I.K. Ge W. Bentley M.A. Paps J. Kramer H.B. Lee J. Aik W. Choi H. Paulsen S.M. Bowman L.A. Loik N.D. Horita S. Ho C.H. Kershaw N.J. et al.Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.Proc. Natl. Acad. Sci. U.S.A. 2014; 111 (25197067): 13331-1333610.1073/pnas.1409916111Crossref PubMed Scopus (49) Google Scholar). For this study we used the PPHD knockout mutant PAO310 to determine its influence on virulence and antibiotic resistance. PAO310 was more virulent than the WT (PAO1) in the G. mellonella model (Fig. 1). This is consistent with a previous study showing higher virulence of PAO310 in a mouse pneumonia model (16Dickinson R.S. Murphy F. Doherty C. Williams S. Mirchandani A. Willson J. Scotti J.S. Preston G. Schofield C.J. Whyte M.K.B. Walmsley S.R. Pseudomonas expression of an oxygen sensing prolyl hydroxylase homologue regulates neutrophil host responses in vitroin vivo.Wellcome Open Res. 2017; 2 (29387803): 10410.12688/wellcomeopenres.12871.1Crossref PubMed Scopus (6) Google Scholar). Although this result may appear contradictory to the previous observation that hypoxia reduces virulence in P. aeruginosa, this is not necessarily the case. If one assumes that PPHD acts in a similar manner to the PHDs in eukaryotic cells (i.e. to repress the effector (HIF) in normoxia), then knocking out the PPHD should indeed mimic hypoxia. However, if in prokaryotes, PPHD activates an antivirulence effector in normoxia, then knocking out PPHD would have the opposite effect to hypoxia (i.e. to increase pathogenicity). Although this is conjuncture at this stage, the full answer to this complex question will require knowledge of the biological roles of PPHD targets in P. aeruginosa and how their hydroxylation affects pathogenicity and antibiotic resistance. Our current studies aim to identify the target(s) for PPHD in P. aeruginosa, and we hope in the future to be able to answer this vitally important question. Resistance to tetracycline antibiotics was selectively increased in PAO310. This was associated with a significant increase in mexE mRNA (Table 1 and Figure 3, Figure 4). MexE is part of the RND efflux pump mexEF–oprN. An increase in tetracycline resistance caused by overexpression of mexEF–oprN was reported before (21Wolloscheck D. Krishnamoorthy G. Nguyen J. Zgurskaya H.I. Kinetic control of quorum sensing in Pseudomonas aeruginosa by multidrug efflux pumps.ACS Infect. Dis. 2018; 4 (29115136): 185-19510.1021/acsinfecdis.7b00160Crossref PubMed Scopus (26) Google Scholar), although it should be noted that whether tetracycline is a major substrate of mexEF–oprN remains controversial (22Lister P.D. Wolter D.J. Hanson N.D. Antibacterial-resistant Pseudomonas aeruginosa: clinical impact and complex regulation of chromosomally encoded resistance mechanisms.Clin. Microbiol. Rev. 2009; 22 (19822890): 582-61010.1128/CMR.00040-09Crossref PubMed Scopus (1178) Google Scholar, 23Fernando D.M. Kumar A. Resistance-nodulation-division multidrug efflux pumps in Gram-negative bacteria: role in virulence.Antibiotics. 2013; 2 (27029297): 163-18110.3390/antibiotics2010163Crossref Scopus (70) Google Scholar). We conclude that PPHD influences efflux pump expression and thus changes antibiotic susceptibility of P. aeruginosa for tetracycline antibiotics. PHD2, the mammalian homolog of PPHD is a key oxygen sensor linked to the HIF pathway (24Pickel C. Taylor C.T. Scholz C.C. Genetic knockdown and pharmacologic inhibition of hypoxia-inducible factor (HIF) hydroxylases.Methods Mol. Biol. 2018; 1742 (29330785): 1-1410.1007/978-1-4939-7665-2_1Crossref PubMed Scopus (4) Google Scholar). We tested whether PPHD inhibition by hypoxia in WT P. aeruginosa (PAO1) has influence on antibiotic susceptibility toward tetracycline antibiotics in a similar way to PPHD knockout. Resistance to tetracycline antibiotics was 2-fold higher in PAO1 at 1% O2 when compared with 21% O2 (Fig. 5). This is similar to the effect seen when PPHD is absent, albeit to a lesser extent. In conclusion, we provide evidence that PPHD activity decreases virulence of P. aeruginosa and increases antibiotic susceptibility to tetracycline antibiotics by altering the expression of the RND efflux pumps mexEF–oprN and MexXY. Therefore, P. aeruginosa PPHD may represent a new therapeutic target in reducing virulence and antibiotic resistance in P. aeruginosa infection. Because PPHD mutation correlates with some of the effects we have reported hypoxia to elicit in P. aeruginosa, these data raise the intriguing possibility that PPHD may provide a molecular link between tissue oxygen levels and bacterial pathogenicity in infectious disease. However, further evidence is required to assess both the oxygen-sensing activity of PPHD and its molecular targets in linking microenvironmental oxygen levels and bacterial pathogenesis and antibiotic resistance. Sixth-instar larvae of G. mellonella (Livefoods direct) were used for the in vivo virulence determination. Bacteria were grown to an A600 of 0.6 and diluted to A600 of 0.1 in PBS (Fisher Chemical). Serial dilutions up to 10−7 in PBS were prepared and plated on agar plates in triplicates to determine the bioburden. 20 μl of each dilution was injected into the hemocoel through the last pro leg (BD microfine U100 insulin syringe; Becton, Dickinson and Company). For each dilution 10 larvae were used and incubated in Petri dishes (Greiner Bio-One) on filter paper (Fisher). 10 larvae were injected with PBS as control. After 24-h incubation, larval death was assessed by the lack of movement upon stimulation. At least three independent experiments were conducted. The data are presented as LD50 values. PAO310 was extracted from a saturating library of sequence-defined transposon insertion mutants. This approach generated over 30,000 defined P. aeruginosa mutants, of which PAO310 was one (28Jacobs M.A. Alwood A. Thaipisuttikul I. Spencer D. Haugen E. Ernst S. Will O. Kaul R. Raymond C. Levy R. Chun-Rong L. Guenthner D. Bovee D. Olson M.V. Manoil C. Comprehensive transposon mutant library of Pseudomonas aeruginosa.Proc. Natl. Acad. Sci. U.S.A. 2003; 100 (14617778): 14339-1434410.1073/pnas.2036282100Crossref PubMed Scopus (848) Google Scholar). To our knowledge, only PPHD is different between PAO310 and PAO1. P. aeruginosa was grown in Luria broth (Sigma) or on tryptic soy agar (Sigma). For hypoxic culture, broth was preincubated at 1% oxygen in a hypoxia chamber (Coy Laboratory) prior to inoculation. Motility was assessed by investigating swimming and swarming. Motility plates were inoculated with a sterile pipette tip (1–10 μl; Star-lab) in the center. After 24 h of incubation at 37 °C, the zone of motility was measured. 10-cm plates with 1% trypthone (Foremedium), 0.5% NaCl (Fisher chemical), and 0.3% agarose (Sigma) were used for the swimming assay. The plates were wrapped with parafilm to avoid dehydration. For swarming 10-cm plates with 0.5% agar (Sigma), 0.5% glucose (Sigma) in Luria broth (0.8 g/100 ml) were prepared. Overnight cultures were diluted to A600 of 0.05. Per condition 1 ml of diluted bacteria suspension was incubated in duplicate in a 12-well plate (Greiner Bio-One) for 24 h. For quantification the biofilm was washed two times with 1 ml of PBS, stained with 0.5 ml of 0.1% crystal violet solution (Sigma) for 15 min at room temperature, and washed again three times with 1 ml of PBS. Crystal violet stain was eluted from biofilm bacteria with 1 ml of ethanol (96%, v/v, Sigma), and absorbance at 595 nm was measured with the CLARIO star spectrometer (BMG Labtech). Dishes with medium only were treated the same way and served as negative control. MIC of P. aeruginosa was determined with several methods. For MIC testing with the VITEK 2 system (Bio Merieux), AST-N352 cards were used according to the manufacturer's instruction. E-test (Bio Merieux) were used according to the manufacturer's instructions. Micro broth dilutions were performed using Sensititre GNX2F susceptibility plate (Thermo Scientific) and with freshly prepared plates using tetracycline hydrochloride (Sigma), doxycycline hydrochloride (Sigma), minocycline hydrochloride (Sigma), and tigecycline hydrate (Sigma). Therefore, serial dilutions of the respective antibiotic in cation adjusted Mueller Hinton broth (Becton, Dickinson and Company) double-concentrated were prepared, and 50 μl were transferred into a 96-well plate (Greiner Bio-One). P. aeruginosa from a freshly grown tryptic soy agar plate were used to prepare a suspension with the same turbidity as a 0.5 McFarland standard. 100 μl of this suspension was added to 11.5 ml of Mueller Hinton broth (Becton, Dickinson and Company). 50 μl were added to the wells with the double-concentrated antibiotics and incubated for 18–24 h at 37 °C. Broth without antibiotic served as growth control. To determine the influence of RND efflux pumps on antibiotic susceptibility, Phe–Arg–β-naphtylamide dihydrochloride (20 μg/ml, Sigma) was added. Furthermore, antibiotic susceptibility testing was performed at 1% oxygen. DNA was extracted from overnight cultures with the Wizard genomic DNA purification kit (Promega) according to the manufacturer's instructions. For RNA extraction overnight cultures of P. aeruginosa were diluted to an A600 of 0.001 and grown statically for 24 h. The bacteria were harvested and processed as described elsewhere (25Hickey C. Schaible B. Nguyen S. Hurley D. Srikumar S. Fanning S. Brown E. Crifo B. Matallanas D. McClean S. Taylor C.T. Schaffer K. Increased virulence of bloodstream over peripheral isolates of P. aeruginosa identified through post-transcriptional regulation of virulence factors.Front. Cell. Infect. Microbiol. 2018; 8 (30416988): 35710.3389/fcimb.2018.00357Crossref PubMed Scopus (10) Google Scholar). 1 μg of RNA was reversed transcribed to cDNA with Superscript II (Invitrogen). To test for the presence of the PPHD gene, PPHD was amplified of genomic DNA with the following primer: forward, 5′-TGA AAA ACG GCC AGT AGC GCG CAT TGA TAC TCC TT-3′, and reverse, 5′-CAG GAA ACA GCT ATG ACC CAC GAT CAA GGT CTG GGG TC-3′. 16S rRNA was amplified as control. Primers for 16S were published previously (26Pérez-Osorio A.C. Williamson K.S. Franklin M.J. Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA (rRNA gene) ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection.J. Bacteriol. 2010; 192 (20348255): 2991-300010.1128/JB.01598-09Crossref PubMed Scopus (69) Google Scholar). To determine gene expression levels, quantitative PCR was carried out as described elsewhere (25Hickey C. Schaible B. Nguyen S. Hurley D. Srikumar S. Fanning S. Brown E. Crifo B. Matallanas D. McClean S. Taylor C.T. Schaffer K. Increased virulence of bloodstream over peripheral isolates of P. aeruginosa identified through post-transcriptional regulation of virulence factors.Front. Cell. Infect. Microbiol. 2018; 8 (30416988): 35710.3389/fcimb.2018.00357Crossref PubMed Scopus (10) Google Scholar). Genes were normalized to P. aeruginosa 16S rRNA. Primers for 16S and mexE were published previously (26Pérez-Osorio A.C. Williamson K.S. Franklin M.J. Heterogeneous rpoS and rhlR mRNA levels and 16S rRNA/rDNA (rRNA gene) ratios within Pseudomonas aeruginosa biofilms, sampled by laser capture microdissection.J. Bacteriol. 2010; 192 (20348255): 2991-300010.1128/JB.01598-09Crossref PubMed Scopus (69) Google Scholar, 27Dumas J.L. van Delden C. Perron K. Köhler T. Analysis of antibiotic resistance gene expression in Pseudomonas aeruginosa by quantitative real-time-PCR.FEMS Microbiol. Lett. 2006; 254 (16445748): 217-22510.1111/j.1574-6968.2005.00008.xCrossref PubMed Scopus (174) Google Scholar). MexX primer were designed with primer 3 (forward, 5′-TGTTCCAGATCGACCCTGC-3′; and reverse, 5′-CTTCGGTGTACTCGCGTTC-3′). The data are presented as means ± S.E. for parametric data and as median for nonparametric data for at least three independent experiments. Statistical analysis was carried out with two-tailed unpaired Student's t test, one-way analysis of variance with Tukey's multiple comparison test, the Mann Whitney test or the Kruskal–Wallis method with Dunne's multiple comparison test. p values of <0.05 were considered statistically significant. B.S., K.S., and C.T.T. conceptualization; B.S., B.C., and C.T.T. data curation; B.S., K.S., and C.T.T. formal analysis; B.S., K.S., and C.T.T. supervision; B.S. and C.T.T. funding acquisition; B.S., B.C., and C.T.T. validation; B.S. and B.C. investigation; B.S. and C.T.T. visualization; B.S. and B.C. methodology; B.S. writing-original draft; K.S. and C.T.T. project administration; K.S. and C.T.T. writing-review and editing; C.T.T. resources. We thank Christopher J. Schoefield and Gail Preston for the PAO310 strain. Download .zip (.14 MB) Help with zip files