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Title: Microevolution in response to transient heme-iron restriction enhances intracellular bacterial community development and persistence}
Article:
Abstract:
Bacterial pathogens must sense, respond and adapt to a myriad of dynamic microenvironmental stressors to survive. Adaptation is key for colonization and long-term ability to endure fluctuations in nutrient availability and inflammatory processes. We hypothesize that strains adapted to survive nutrient deprivation are more adept for colonization and establishment of chronic infection. In this study, we detected microevolution in response to transient nutrient limitation through mutation of icc. The mutation results in decreased 3', 5'-cyclic adenosine monophosphate phosphodiesterase activity in nontypeable Haemophilus influenzae( NTHI). In a preclinical model of NTHI-induced otitis media( OM), we observed a significant decrease in the recovery of effusion from ears infected with the icc mutant strain. Clinically, resolution of OM coincides with the clearance of middle ear fluid. In contrast to this clinical paradigm, we observed that the icc mutant strain formed significantly more intracellular bacterial communities( IBCs) than the parental strain early during experimental OM. Although the number of IBCs formed by the parental strain was low at early stages of OM, we observed a significant increase at later stages that coincided with absence of recoverable effusion, suggesting the presence of a mucosal reservoir following resolution of clinical disease. These data provide the first insight into NTHI microevolution during nutritional limitation and provide the first demonstration of IBCs in a preclinical model of chronic OM.
Introduction:
The host sequesters essential micronutrients( i. e. metals) as a mechanism to limit bacterial infection[1]. Bacteria that require exogenous nutrients for growth are subjected to stresses that necessitate adaptation for survival. Bacteria can adapt to these stresses through transcriptional, epigenetic and genetic mechanisms[1]. Identification of genes subject to microevolution provides insight into the nutritional and environmental stresses incurred during infection as well as new therapeutic targets to quell infection. Nontypeable Haemophilus influenzae( NTHI) is a heme auxotroph and must obtain this essential nutrient from the environment[2]. The presence of heme-containing compounds is sufficient to support polymicrobial growth in the nasopharynx[3]. During ascent from the nasopharynx to the middle ear, NTHI experiences fluctuations in nutrient availability, particularly heme-iron. The lack of sufficient heme-iron in the middle ear is indicated by the increase in the number of iron-uptake genes in NTHI strains isolated from children with otitis media[( OM);[4]]. Furthermore, iron-uptake genes are transcribed in NTHI early during experimental OM[5, 6]. We previously demonstrated that transient heme-iron restriction of NTHI potentiates changes in bacterial morphology, biofilm architecture, disease severity and provides a survival advantage in the preclinical model of OM[7]. NTHI inhabits diverse niches in the host including the nasopharynx, middle ear, sinus, eye and lung. At these sites, NTHI can be planktonic, in a biofilm or associated with mucosa[8]. NTHI has also been observed within intracellular niches in vitro in cultured lung and middle ear epithelial cells, as well as in clinical biopsies of adenoid and middle ear tissues from children with a history of OM[7, 9–12]. New paradigms for recurrent infections were revealed through the identification of intracellular bacterial communities( IBCs) in the cytoplasm of epithelial cells. This complex intracellular lifestyle, first described for uropathogenic Escherichia coli[( UPEC);[13]], provides access to nutrients and protection from antibiotic therapies and innate immune effectors[14, 15]. The IBC pathway is critical for acute urinary tract infection and culminates in a latent intracellular population that serves as a bona fide reservoir for recurrent infection[15]. Although first described in preclinical models, IBCs are present in bladder biopsies and in the urine of patients with urinary tract infections caused by UPEC[13]. IBCs of Klebsiella and Proteus have also been observed in the urinary tract[16, 17] and Helicobacter pylori in the stomach[18], in preclinical models of urinary tract infection and gastritis, respectively. We recently observed the formation of NTHI IBCs in cultured chinchilla middle ear epithelial cells in response to transient heme-iron limitation[7]. This observation suggests a potential role for intracellular populations of NTHI during OM and reveals new potential mechanisms for disease chronicity, persistence and recurrence that remain underexplored. The ability to adapt to new microenvironments is consistent with the predominance of NTHI as a causative agent of OM, the most common bacterial infection in children[19–21]. Longitudinal sampling of patients with chronic obstructive pulmonary disease( COPD) revealed the same NTHI strains, despite intermittent periods of negative culture[22]. In this study, we evaluate the microevolution of NTHI associated with adaptation and persistence in response to nutrient limitation. In long-term stationary phase following transient heme-iron restriction, we observed a single nucleotide polymorphism in icc that abolishes 3’, 5’-cyclic adenosine monophosphate( cAMP) phosphodiesterase activity. In the preclinical model for NTHI-induced OM, the icc mutant significantly shifts infection kinetics, with decreased middle ear fluid, a clinical indicator of disease resolution. However, the icc mutant was still associated with middle ear tissues at levels similar to the parental strain, suggesting that the presence of fluid and bacterial burden of the middle ear can be uncoupled. Although the bacterial burdens were similar, there was a significant increase in IBC formation by the icc mutant strain early during disease while formation of IBCs by the parental strain occurred gradually, indicating that NTHI adaptation to an intracellular niche occurs during active infection. Using UPEC as the paradigm, this observation suggests that NTHI intracellular populations may serve as a reservoir and contribute to both chronic and recurrent disease associated with OM.
Discussion:
The diversity of NTHI lifestyles expands beyond commensalism in the nasopharynx and biofilm formation in the middle ear, to now include intracellular populations of bacteria, a phenotype known to contribute to persistence and recurrence in UPEC, Proteus, Klebsiella and Helicobacter[15]. In vitro models have revealed the potential importance of intracellular NTHI, but in these models NTHI typically traffics through the endolysosomal pathway[11, 33, 35]. We have previously demonstrated that transient heme-iron restriction promotes IBC formation in cultured middle ear epithelial cells in vitro[7]. Here, we now provide evidence that NTHI completely fills the cytoplasm of middle ear epithelial cells during experimental OM. Thus far, IBCs have primarily been attributed to pathogens of the urinary tract, but our studies now reveal IBCs formed by a pathogen of the respiratory tract. Thus, our studies expand upon previous in vitro models and use a preclinical model of OM to provide mechanistic insight into an understudied intracellular niche and the potential contribution to persistence and recurrence. IBCs formed by UPEC and other species confer tolerance to antibiotics, as well as protection from innate and adaptive immune responses[14, 15]. Further, the intracellular environment provides access to nutrients that are limited in the urine[16, 36, 37]. IBCs are critical for persistence and recurrence for urinary tract infection[15]. These studies have shifted the clinical perspective from a focus on recurrence due to reinoculation to include recurrences from an intracellular bladder reservoir. The implications of these findings have already impacted approaches to treat and prevent recurrent infection[38, 39]. Based upon the UPEC paradigm that IBCs lead to an intrabladder reservoir for reinfection, it is of interest to speculate that IBCs in the middle ear provide a viable population of bacteria to initiate recurrent OM in the absence of reinoculation. NTHI was recovered from middle ear aspirates from 57% of children with persistent OM at all visits during a longitudinal study[40]. Furthermore, the same strain of NTHI was isolated from the same COPD patient in a longitudinal study, despite periods of negative culture[22]. As such, the observed persistence of NTHI at multiple niches requires new approaches to prevent and treat infection. Persistent bacterial populations can arise stochastically, but also as a result of stressors that include, among others, nutrient limitation[41–45]. The use of heme-iron restriction as a nutritional stressor dramatically increases the longevity of NTHI in stationary phase cultures( Figs 1 and 2). In a preclinical model of OM, the strain adapted though transient heme-iron restriction( RM33) exhibited increased IBC formation as compared with the parent, suggesting that adaptation to fluctuations in nutrient availability promotes an intracellular lifestyle. Our discovery of IBCs in vivo combined with the ability of NTHI to form IBCs in cultured epithelial cells allows investigation of differential trafficking of bacteria either to the lysosome or for productive IBC formation. In our study we focused on nutrient limitation, but the complexity of the host environment suggests that other immune stressors may contribute to NTHI microevolution during disease. Ongoing investigations will delineate specific bacterial factors and mechanisms of invasion that promote IBC formation. In two independent cultures, we observed microevolution of icc which encodes the only cAMP phosphodiesterase in Haemophilus. Icc degrades cAMP to modulate the intracellular concentration of this important signaling molecule. DNA import is regulated by cytoplasmic cAMP levels[30] and we and others have shown that defects in cAMP phosphodiesterase increases competence[Fig 4D;[30]]. Bacteria can utilize exogenous DNA for horizontal gene transfer, DNA repair, or as a nutritional source[46, 47]. Use as a nutritional source is a compelling concept and is supported by the observations that DNA is readily available in host niches and only 10–15% of the DNA taken up by NTHI is incorporated intact into the genome, the remainder is degraded[48–51]. The link between nutrition, cAMP levels and competence is supported by the requirement for nutrient limitation as an inducer of competence in Haemophilus[52]. Moreover, nutrient availability signals catabolite repression to control levels of cAMP through the enzymatic activities of adenylate cyclase and cAMP phosphodiesterase combined with the cAMP receptor protein( CRP)[53, 54]. In addition to the canonical CRP binding site in E. coli( CRP-N), Haemophilus has an additional regulatory site( CRP-S) that uses Sxy for the regulation of competence[55–58]. Thus, there is overlap between CRP-N and CRP-S directed regulation and a close link between sensing nutritional status and regulation of competence in H. influenzae[59]. Although we used competence as a tool to determine changes in the levels of cAMP in the adapted strain( RM33), the contribution of competence or any other member of the CRP regulon in the phenotypes observed during infection remain to be elucidated. These studies provide a new model to delineate the role of cAMP in the development of IBCs. In our study, we observe a decrease in cAMP phosphodiesterase activity following nutrient restriction with an increase in persistence, consistent with the role of cAMP and catabolite repression in nutrient limited conditions. In contrast, other studies provide evidence that a decrease in cAMP levels is associated with increased antibiotic-mediated persister cell formation[60, 61]. Thus, the levels of cAMP appear to modulate persistence through multiple mechanisms based upon the type of stressor encountered. Icc is likely not the only target for adaptation in vivo. During disease, fluctuation in nutrients as well as immune stressors may influence adaptation through direct modulation of cAMP levels, indirectly through mutation or regulation of other members of the cAMP regulon, or other as yet unidentified processes. NTHI was not recovered from the mucosal tissues of some ears( Fig 5D), yet we observed IBCs in all sections evaluated( Fig 6C), suggesting that NTHI within IBCs may not be liberated using standard homogenization techniques or that this population is viable but not culturable following intracellular growth. It should be noted that the IBCs counted represent the number present in a 4μm thin section, which suggests that our quantitation will underestimate the magnitude of intracellular NTHI. In fact, recent studies have demonstrated that enumeration of viable bacteria does not accurately represent the number of bacteria within IBCs[62]. Recent studies have also determined that UPEC IBCs are metabolically active in the bladder[63]. Future studies will evaluate the metabolic activity of NTHI in IBCs in the middle ear. Collectively, our observations reveal microevolution in response to nutrient limitation leading to persistence in long-term culture, and that this adapted strain exhibited a predilection for productive growth within the epithelial environment which resulted in increased IBC formation during experimental OM. Perhaps even more interesting, we observed an increase in IBC formation of the parent at 28 days during experimental OM, suggesting that genotypic or phenotypic adaptation occurs during infection. As the availability of nutrients change due to the dynamic immune response, we speculate that the intracellular environment provides nutrients leading to bacterial proliferation and IBC formation. Moreover, the presence of IBCs during OM provides the first opportunity to evaluate the role of intracellular populations in chronicity and quiescence as a new paradigm for recurrent OM. This model provides a new platform for the identification of novel diagnostic and therapeutic approaches for OM. Our prior proteomic studies demonstrate an increase in the actin filament branching protein, Arp2/3, during acute experimental OM and that inhibition of Arp2/3 decreases NTHI invasion as a potential adjunct therapy[64]. Finally, it is of interest to determine whether intracellular populations contribute to the other NTHI-mediated diseases such as conjunctivitis, sinusitis, pneumonia, and exacerbations in COPD and cystic fibrosis[65, 66].
Nontypeable Haemophilus influenzae ( NTHI ) inhabits diverse niches in the host . The ability to adapt to new microenvironments is consistent with the predominance of NTHI as a causative agent of otitis media ( OM ) in children . We evaluated the microevolution of NTHI associated with adaptation and persistence in response to nutrient limitation . We identified a naturally occurring mutation that enhances NTHI persistence and formation of intracellular bacterial communities ( IBCs ) in a pre-clinical model of OM . The presence of IBCs during OM provides the first opportunity to evaluate the role of intracellular populations in chronicity and quiescence as a new paradigm for recurrent OM . This model provides a new platform to identify novel therapeutics for this highly prevalent and costly infectious disease .