Conserved metabolic regulator ArcA responds to oxygen availability, iron limitation, and cell envelope perturbations during bacteremia.

IMPORTANCE: Infections of the bloodstream are life-threatening and can result in sepsis. Gram-negative bacteria cause a significant portion of bloodstream infections, which is also referred to as bacteremia. The long-term goal of our work is to understand how such bacteria establish and maintain infection during bacteremia. We have previously identified the transcription factor ArcA, which promotes fermentation in bacteria, as a likely contributor to the growth and survival of bacteria in this environment. Here, we study ArcA in the Gram-negative species Citrobacter freundii, Klebsiella pneumoniae, and Serratia marcescens. Our findings aid in determining how these bacteria sense their environment, utilize nutrients, and generate energy while countering the host immune system. This information is critical for developing better models of infection to inform future therapeutic development.

The ArcAB Two-Component System: Function in Metabolism, Redox Control, and Infection.

ArcAB, also known as the Arc system, is a member of the two-component system family of bacterial transcriptional regulators and is composed of sensor kinase ArcB and response regulator ArcA. In this review, we describe the structure and function of these proteins and assess the state of the literature regarding ArcAB as a sensor of oxygen consumption. The bacterial quinone pool is the primary modulator of ArcAB activity, but questions remain for how this regulation occurs. This review highlights the role of quinones and their oxidation state in activating and deactivating ArcB and compares competing models of the regulatory mechanism. The cellular processes linked to ArcAB regulation of central metabolic pathways and potential interactions of the Arc system with other regulatory systems are also reviewed. Recent evidence for the function of ArcAB under aerobic conditions is challenging the long-standing characterization of this system as strictly an anaerobic global regulator, and the support for additional ArcAB functionality in this context is explored. Lastly, ArcAB-controlled cellular processes with relevance to infection are assessed.

Identification of distinct capsule types associated with Serratia marcescens infection isolates.

Serratia marcescens is a versatile opportunistic pathogen that can cause a variety of infections, including bacteremia. Our previous work established that the capsule polysaccharide (CPS) biosynthesis and translocation locus contributes to the survival of S. marcescens in a murine model of bacteremia and in human serum. In this study, we determined the degree of capsule genetic diversity among S. marcescens isolates. Capsule loci (KL) were extracted from >300 S. marcescens genome sequences and compared. A phylogenetic comparison of KL sequences demonstrated a substantial level of KL diversity within S. marcescens as a species and a strong delineation between KL sequences originating from infection isolates versus environmental isolates. Strains from five of the identified KL types were selected for further study and electrophoretic analysis of purified CPS indicated the production of distinct glycans. Polysaccharide composition analysis confirmed this observation and identified the constituent monosaccharides for each strain. Two predominant infection-associated clades, designated KL1 and KL2, emerged from the capsule phylogeny. Bacteremia strains from KL1 and KL2 were determined to produce ketodeoxynonulonic acid and N-acetylneuraminic acid, two sialic acids that were not found in strains from other clades. Further investigation of KL1 and KL2 sequences identified two genes, designated neuA and neuB, that were hypothesized to encode sialic acid biosynthesis functions. Disruption of neuB in a KL1 isolate resulted in the loss of sialic acid and CPS production. The absence of sialic acid and CPS production also led to increased susceptibility to internalization by a human monocytic cell line, demonstrating that S. marcescens phagocytosis resistance requires CPS. Together, these results establish the capsule genetic repertoire of S. marcescens and identify infection-associated clades with sialic acid CPS components.

Replication Dynamics for Six Gram-Negative Bacterial Species during Bloodstream Infection.

Bloodstream infections (BSI) are a major public health burden due to high mortality rates and the cost of treatment. The impact of BSI is further compounded by a rise in antibiotic resistance among Gram-negative species associated with these infections. Escherichia coli, Serratia marcescens, Klebsiella pneumoniae, Enterobacter hormaechei, Citrobacter freundii, and Acinetobacter baumannii are all common causes of BSI, which can be recapitulated in a murine model. The objective of this study was to characterize infection kinetics and bacterial replication rates during bacteremia for these six pathogens to gain a better understanding of bacterial physiology during infection. Temporal observations of bacterial burdens of the tested species demonstrated varied abilities to establish colonization in the spleen, liver, or kidney. K. pneumoniae and S. marcescens expanded rapidly in the liver and kidney, respectively. Other organisms, such as C. freundii and E. hormaechei, were steadily cleared from all three target organs throughout the infection. In situ replication rates measured by whole-genome sequencing of bacterial DNA recovered from murine spleens demonstrated that each species was capable of sustained replication at 24 h postinfection, and several species demonstrated <60-min generation times. The relatively short generation times observed in the spleen were in contrast to an overall decrease in bacterial burden for some species, suggesting that the rate of immune-mediated clearance exceeded replication. Furthermore, bacterial generation times measured in the murine spleen approximated those measured during growth in human serum cultures. Together, these findings provide insight into the infection kinetics of six medically important species during bacteremia. IMPORTANCE Bloodstream infections are a global public health problem. The goal of this work was to determine the replication characteristics of Gram-negative bacterial species in the host following bloodstream infection. The number of bacteria in major organs is likely determined by a balance between replication rates and the ability of the host to clear bacteria. We selected a cohort of six species from three families that represent common causative agents of bloodstream infections in humans and determined their replication rates in a murine bacteremia model. We found that the bacteria grow rapidly in the spleen, demonstrating that they can obtain the necessary nutrients for growth in this environment. However, the overall number of bacteria decreased in most cases, suggesting that killing of bacteria outpaces their growth. Through a better understanding of how bacteria replicate during bloodstream infections, we aim to gain insight into future means of combating these infections.

Pathogenesis of Gram-Negative Bacteremia.

Gram-negative bacteremia is a devastating public health threat, with high mortality in vulnerable populations and significant costs to the global economy. Concerningly, rates of both Gram-negative bacteremia and antimicrobial resistance in the causative species are increasing. Gram-negative bacteremia develops in three phases. First, bacteria invade or colonize initial sites of infection. Second, bacteria overcome host barriers, such as immune responses, and disseminate from initial body sites to the bloodstream. Third, bacteria adapt to survive in the blood and blood-filtering organs. To develop new therapies, it is critical to define species-specific and multispecies fitness factors required for bacteremia in model systems that are relevant to human infection. A small subset of species is responsible for the majority of Gram-negative bacteremia cases, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii The few bacteremia fitness factors identified in these prominent Gram-negative species demonstrate shared and unique pathogenic mechanisms at each phase of bacteremia progression. Capsule production, adhesins, and metabolic flexibility are common mediators, whereas only some species utilize toxins. This review provides an overview of Gram-negative bacteremia, compares animal models for bacteremia, and discusses prevalent Gram-negative bacteremia species.

Sulfur Assimilation Alters Flagellar Function and Modulates the Gene Expression Landscape of Serratia marcescens.

Sulfur is an essential nutrient that contributes to cellular redox homeostasis, transcriptional regulation, and translation initiation when incorporated into different biomolecules. Transport and reduction of extracellular sulfate followed by cysteine biosynthesis is a major pathway of bacterial sulfur assimilation. For the opportunistic pathogen Serratia marcescens, function of the cysteine biosynthesis pathway is required for extracellular phospholipase activity and flagellum-mediated surface motility, but little else is known about the influence of sulfur assimilation on the physiology of this organism. In this work, it was determined that an S. marcescens cysteine auxotroph fails to differentiate into hyperflagellated and elongated swarmer cells and that cysteine, but not other organic sulfur molecules, restores swarming motility to these bacteria. The S. marcescens cysteine auxotroph further exhibits reduced transcription of phospholipase, hemolysin, and flagellin genes, each of which is subject to transcriptional control by the flagellar regulatory system. Based on these data and the central role of cysteine in sulfur assimilation, it was reasoned that environmental sulfur availability may contribute to the regulation of these functions in S. marcescens Indeed, bacteria that are starved for sulfate exhibit substantially reduced transcription of the genes for hemolysin, phospholipase, and the FlhD flagellar master regulator. A global transcriptomic analysis further defined a large set of S. marcescens genes that are responsive to extracellular sulfate availability, including genes that encode membrane transport, nutrient utilization, and metabolism functions. Finally, sulfate availability was demonstrated to alter S. marcescens cytolytic activity, suggesting that sulfate assimilation may impact the virulence of this organism.IMPORTANCE Serratia marcescens is a versatile bacterial species that inhabits diverse environmental niches and is capable of pathogenic interactions with host organisms ranging from insects to humans. This report demonstrates for the first time the extensive impacts that environmental sulfate availability and cysteine biosynthesis have on the transcriptome of S. marcescens The finding that greater than 1,000 S. marcescens genes are differentially expressed depending on sulfate availability suggests that sulfur abundance is a crucial factor that controls the physiology of this organism. Furthermore, the high relative expression levels for the putative virulence factors flagella, phospholipase, and hemolysin in the presence of sulfate suggests that a sulfur-rich host environment could contribute to the transcription of these genes during infection.

Twin arginine translocation, ammonia incorporation, and polyamine biosynthesis are crucial for Proteus mirabilis fitness during bloodstream infection.

The Gram-negative bacterium Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTI), which can progress to secondary bacteremia. While numerous studies have investigated experimental infection with P. mirabilis in the urinary tract, little is known about pathogenesis in the bloodstream. This study identifies the genes that are important for survival in the bloodstream using a whole-genome transposon insertion-site sequencing (Tn-Seq) approach. A library of 50,000 transposon mutants was utilized to assess the relative contribution of each non-essential gene in the P. mirabilis HI4320 genome to fitness in the livers and spleens of mice at 24 hours following tail vein inoculation compared to growth in RPMI, heat-inactivated (HI) naïve serum, and HI acute phase serum. 138 genes were identified as ex vivo fitness factors in serum, which were primarily involved in amino acid transport and metabolism, and 143 genes were identified as infection-specific in vivo fitness factors for both spleen and liver colonization. Infection-specific fitness factors included genes involved in twin arginine translocation, ammonia incorporation, and polyamine biosynthesis. Mutants in sixteen genes were constructed to validate both the ex vivo and in vivo results of the transposon screen, and 12/16 (75%) exhibited the predicted phenotype. Our studies indicate a role for the twin arginine translocation (tatAC) system in motility, translocation of potential virulence factors, and fitness within the bloodstream. We also demonstrate the interplay between two nitrogen assimilation pathways in the bloodstream, providing evidence that the GS-GOGAT system may be preferentially utilized. Furthermore, we show that a dual-function arginine decarboxylase (speA) is important for fitness within the bloodstream due to its role in putrescine biosynthesis rather than its contribution to maintenance of membrane potential. This study therefore provides insight into pathways needed for fitness within the bloodstream, which may guide strategies to reduce bacteremia-associated mortality.

Citrobacter freundii fitness during bloodstream infection.

Sepsis resulting from microbial colonization of the bloodstream is a serious health concern associated with high mortality rates. The objective of this study was to define the physiologic requirements of Citrobacter freundii in the bloodstream as a model for bacteremia caused by opportunistic Gram-negative pathogens. A genetic screen in a murine host identified 177 genes that contributed significantly to fitness, the majority of which were broadly classified as having metabolic or cellular maintenance functions. Among the pathways examined, the Tat protein secretion system conferred the single largest fitness contribution during competition infections and a putative Tat-secreted protein, SufI, was also identified as a fitness factor. Additional work was focused on identifying relevant metabolic pathways for bacteria in the bloodstream environment. Mutations that eliminated the use of glucose or mannitol as carbon sources in vitro resulted in loss of fitness in the murine model and similar results were obtained upon disruption of the cysteine biosynthetic pathway. Finally, the conservation of identified fitness factors was compared within a cohort of Citrobacter bloodstream isolates and between Citrobacter and Serratia marcescens, the results of which suggest the presence of conserved strategies for bacterial survival and replication in the bloodstream environment.

TnseqDiff: identification of conditionally essential genes in transposon sequencing studies.

BACKGROUND: Tn-Seq is a high throughput technique for analysis of transposon mutant libraries to determine conditional essentiality of a gene under an experimental condition. A special feature of the Tn-seq data is that multiple mutants in a gene provides independent evidence to prioritize that gene as being essential. The existing methods do not account for this feature or rely on a high-density transposon library. Moreover, these methods are unable to accommodate complex designs. RESULTS: The method proposed here is specifically designed for the analysis of Tn-Seq data. It utilizes two steps to estimate the conditional essentiality for each gene in the genome. First, it collects evidence of conditional essentiality for each insertion by comparing read counts of that insertion between conditions. Second, it combines insertion-level evidence for the corresponding gene. It deals with data from both low- and high-density transposon libraries and accommodates complex designs. Moreover, it is very fast to implement. The performance of the proposed method was tested on simulated data and experimental Tn-Seq data from Serratia marcescens transposon mutant library used to identify genes that contribute to fitness in a murine model of infection. CONCLUSION: We describe a new, efficient method for identifying conditionally essential genes in Tn-Seq experiments with high detection sensitivity and specificity. It is implemented as TnseqDiff function in R package Tnseq and can be installed from the Comprehensive R Archive Network, CRAN.

Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity.

Serratia marcescens causes health care-associated opportunistic infections that can be difficult to treat due to a high incidence of antibiotic resistance. One of the many secreted proteins of S. marcescens is the PhlA phospholipase enzyme. Genes involved in the production and secretion of PhlA were identified by screening a transposon insertion library for phospholipase-deficient mutants on phosphatidylcholine-containing medium. Mutations were identified in four genes (cyaA, crp, fliJ, and fliP) that are involved in the flagellum-dependent PhlA secretion pathway. An additional phospholipase-deficient isolate harbored a transposon insertion in the cysE gene encoding a predicted serine O-acetyltransferase required for cysteine biosynthesis. The cysE requirement for extracellular phospholipase activity was confirmed using a fluorogenic phospholipase substrate. Phospholipase activity was restored to the cysE mutant by the addition of exogenous l-cysteine or O-acetylserine to the culture medium and by genetic complementation. Additionally, phlA transcript levels were decreased 6-fold in bacteria lacking cysE and were restored with added cysteine, indicating a role for cysteine-dependent transcriptional regulation of S. marcescens phospholipase activity. S. marcescenscysE mutants also exhibited a defect in swarming motility that was correlated with reduced levels of flhD and fliA flagellar regulator gene transcription. Together, these findings suggest a model in which cysteine is required for the regulation of both extracellular phospholipase activity and surface motility in S. marcescensIMPORTANCESerratia marcescens is known to secrete multiple extracellular enzymes, but PhlA is unusual in that this protein is thought to be exported by the flagellar transport apparatus. In this study, we demonstrate that both extracellular phospholipase activity and flagellar function are dependent on the cysteine biosynthesis pathway. Furthermore, a disruption of cysteine biosynthesis results in decreased phlA and flagellar gene transcription, which can be restored by supplying bacteria with exogenous cysteine. These results identify a previously unrecognized role for CysE and cysteine in the secretion of S. marcescens phospholipase and in bacterial motility.

Capsule Production and Glucose Metabolism Dictate Fitness during Serratia marcescens Bacteremia.

Serratia marcescens is an opportunistic pathogen that causes a range of human infections, including bacteremia, keratitis, wound infections, and urinary tract infections. Compared to other members of the Enterobacteriaceae family, the genetic factors that facilitate Serratia proliferation within the mammalian host are less well defined. An in vivo screen of transposon insertion mutants identified 212 S. marcescens fitness genes that contribute to bacterial survival in a murine model of bloodstream infection. Among those identified, 11 genes were located within an 18-gene cluster encoding predicted extracellular polysaccharide biosynthesis proteins. A mutation in the wzx gene contained within this locus conferred a loss of fitness in competition infections with the wild-type strain and a reduction in extracellular uronic acids correlating with capsule loss. A second gene, pgm, encoding a phosphoglucomutase exhibited similar capsule-deficient phenotypes, linking central glucose metabolism with capsule production and fitness of Serratia during mammalian infection. Further evidence of the importance of central metabolism was obtained with a pfkA glycolytic mutant that demonstrated reduced replication in human serum and during murine infection. An MgtB magnesium transporter homolog was also among the fitness factors identified, and an S. marcescens mgtB mutant exhibited decreased growth in defined medium containing low concentrations of magnesium and was outcompeted ~10-fold by wild-type bacteria in mice. Together, these newly identified genes provide a more complete understanding of the specific requirements for S. marcescens survival in the mammalian host and provide a framework for further investigation of the means by which S. marcescens causes opportunistic infections.IMPORTANCESerratia marcescens is a remarkably prolific organism that replicates in diverse environments, including as an opportunistic pathogen in human bacteremia. The genetic requirements for S. marcescens survival in the mammalian bloodstream were defined in this work by transposon insertion sequencing. In total, 212 genes that contribute to bacterial fitness were identified. When sorted via biological function, two of the major fitness categories identified herein were genes encoding capsule polysaccharide biogenesis functions and genes involved in glucose utilization. Further investigation determined that certain glucose metabolism fitness genes are also important for the generation of extracellular polysaccharides. Together, these results identify critical biological processes that allow S. marcescens to colonize the mammalian bloodstream.

Seminal Plasma Promotes Neisseria gonorrhoeae Aggregation and Biofilm Formation.

UNLABELLED: Neisseria gonorrhoeae causes the human-specific disease gonorrhea and is transmitted from person to person primarily via sexual contact. During transmission, N. gonorrhoeae is often exposed to seminal fluid and must adapt to this change in environment. Previous work demonstrated that seminal fluid facilitates N. gonorrhoeae motility and alters epithelial cell interactions. In this study, exposure to seminal fluid was found to decrease surface adherence of gonococci in a manner that was independent of Opa adhesin proteins or type IV pilus retraction. Semen was also shown to cause dispersal of bacteria that had previously established surface adherence. Although surface adherence decreased, interbacterial interactions were increased by seminal plasma both in long-term static culture and on a cell-to-cell basis over shorter time periods. The result of increased bacterium-bacterium interactions resulted in the formation of microcolonies, an important step in the N. gonorrhoeae infectious process. Seminal fluid also facilitated increased bacterial aggregation in the form of shear-resistant three-dimensional biofilms. These results emphasize the importance of the gonococcal response to the influx of seminal fluid within the genital niche. Further characterization of the N. gonorrhoeae response to semen will advance our understanding of the mechanisms behind the establishment of infection in naive hosts and the process of transmission. IMPORTANCE: N. gonorrhoeae is the causative agent of the globally prevalent sexually transmitted infection gonorrhea. An understudied aspect of this human-adapted pathogen is the change in bacterial physiology that occurs during sexual transmission. N. gonorrhoeae encounters semen when transmitted from host to host, and it is known that, when N. gonorrhoeae is exposed to seminal fluid, alterations in bacterial motility and type IV pilus arrangement occur. This work extends our previous observations on this modulation of gonococcal physiology by seminal fluid and demonstrates that seminal plasma decreases surface adherence, promotes interbacterial interactions, and enhances biofilm formation.

Seminal plasma initiates a Neisseria gonorrhoeae transmission state.

Niche-restricted pathogens are evolutionarily linked with the specific biological fluids that are encountered during infection. Neisseria gonorrhoeae causes the genital infection gonorrhea and is exposed to seminal fluid during sexual transmission. Treatment of N. gonorrhoeae with seminal plasma or purified semen proteins lactoferrin, serum albumin, and prostate-specific antigen each facilitated type IV pilus-mediated twitching motility of the bacterium. Motility in the presence of seminal plasma was characterized by high velocity and low directional persistence. In addition, infection of epithelial cells with N. gonorrhoeae in the presence of seminal plasma resulted in enhanced microcolony formation. Close association of multiple pili in the form of bundles was also disrupted after seminal plasma treatment leading to an increase in the number of single pilus filaments on the bacterial surface. Thus, exposure of N. gonorrhoeae to seminal plasma is proposed to alter bacterial motility and aggregation characteristics to influence the processes of transmission and colonization. IMPORTANCE There are greater than 100 million estimated new cases of gonorrhea annually worldwide. Research characterizing the mechanisms of pathogenesis and transmission of Neisseria gonorrhoeae is important for developing new prevention strategies, since antibiotic resistance of the organism is becoming increasingly prevalent. Our work identifies seminal plasma as a mediator of N. gonorrhoeae twitching motility and microcolony formation through functional modification of the type IV pilus. These findings provide insight into motility dynamics and epithelial cell colonization under conditions that are relevant to sexual transmission. Type IV pili are common virulence factors with diverse functions among bacterial pathogens, and this work identifies interactions between type IV pili and the host environment. Finally, this work illustrates the importance of the host environment and niche-specific fluids on microbial pathogenesis.

Phase variation leads to the misidentification of a Neisseria gonorrhoeae virulence gene.

Neisseria gonorrhoeae is the causative agent of gonorrhea and an obligate pathogen of humans. The Opa proteins of these bacteria are known to mediate attachment and internalization by host cells, including neutrophils. The Opa protein repertoire of a typical N. gonorrhoeae isolate is encoded on ~11 genes distributed throughout the chromosome and is subject to stochastic changes in expression through phase variation. Together, these characteristics make Opa proteins a critical yet unpredictable aspect of any experimental investigation into the interaction of N. gonorrhoeae with host cells. The goal of this study was to identify novel virulence factors of N. gonorrhoeae by assessing the contribution of a set of uncharacterized hydrogen peroxide-induced genes to bacterial survival against neutrophil-mediated killing. To this end, a strain harboring an engineered mutation in the NGO0322 gene was identified that exhibited increased sensitivity to neutrophil-mediated killing, enhanced internalization by neutrophils, and the ability to induce high levels of neutrophil-generated reactive oxygen species. Each of these phenotypes reverted to near wild-type levels following genetic complementation of the NGO0322 mutation. However, after immunoblot analysis of Opa proteins expressed by the isogenic parent, mutant, and genetically complemented strains, it was determined that phase variation had resulted in a disparity between the Opa profiles of these strains. To determine whether Opa phase variation, rather than NGO0322 mutation, was the cause of the observed neutrophil-related phenotypes, NGO0322 function was investigated in N. gonorrhoeae strains lacking all Opa proteins or constitutively expressing the OpaD variant. In both cases, mutation of NGO0322 did not alter survival of gonococci in the presence of neutrophils. These results demonstrate the importance of controlling for the frequent and random variation in Opa protein production by N. gonorrhoeae when investigating host cell interactions.

Neisseria gonorrhoeae and humans perform an evolutionary LINE dance.

Horizontal gene transfer is an important mechanism for generating genetic diversity. As the number of sequenced genomes continues to increase, so do the examples of horizontal genetic exchange between both related and divergent organisms. Here we discuss the recent finding that certain strains of the human pathogen Neisseria gonorrhoeae have incorporated a small fragment of human DNA sequence into their genomes. The horizontally acquired sequence exhibits 98-100% nucleotide identity to a 685 bp portion of the highly repetitive retrotransposable element L1 and its presence in the gonococcal genome has been confirmed by multiple molecular techniques. The possibility of similar L1 horizontal gene transfer events having occurred in other bacteria based on genomic sequence evidence is explored. Potential mechanisms of how N. gonorrhoeae was able to acquire and maintain this human sequence are also discussed in addition to the evolutionary implications of such an event.

Opportunity and means: horizontal gene transfer from the human host to a bacterial pathogen.

The acquisition and incorporation of genetic material between nonmating species, or horizontal gene transfer (HGT), has been frequently described for phylogenetically related organisms, but far less evidence exists for HGT between highly divergent organisms. Here we report the identification and characterization of a horizontally transferred fragment of the human long interspersed nuclear element L1 to the genome of the strictly human pathogen Neisseria gonorrhoeae. A 685-bp sequence exhibiting 98 to 100% identity to copies of the human L1 element was identified adjacent to the irg4 gene in some N. gonorrhoeae genomes. The L1 fragment was observed in ~11% of the N. gonorrhoeae population sampled but was not detected in Neisseria meningitidis or commensal Neisseria isolates. In addition, N. gonorrhoeae transcripts containing the L1 sequence were detected by reverse transcription-PCR, indicating that an L1-derived gene product may be produced. The high degree of identity between human and gonococcal L1 sequences, together with the absence of L1 sequences from related Neisseria species, indicates that this HGT event occurred relatively recently in evolutionary history. The identification of L1 sequences in N. gonorrhoeae demonstrates that HGT can occur between a mammalian host and a resident bacterium, which has important implications for the coevolution of both humans and their associated microorganisms.

Differential expression of Bordetella pertussis iron transport system genes during infection.

Temporal expression patterns of the Bordetella pertussis alcaligin, enterobactin and haem iron acquisition systems were examined using alcA-, bfeA- and bhuR-tnpR recombinase fusion strains in a mouse respiratory infection model. The iron systems were differentially expressed in vivo, showing early induction of the alcaligin and enterobactin siderophore systems, and delayed induction of the haem system in a manner consistent with predicted changes in host iron source availability during infection. Previous mixed infection competition studies established the importance of alcaligin and haem utilization for B. pertussis in vivo growth and survival. In this study, the contribution of the enterobactin system to the fitness of B. pertussis was confirmed using wild-type and enterobactin receptor mutant strains in similar competition infection experiments. As a correlate to the in vivo expression studies of B. pertussis iron systems in mice, sera from uninfected and B. pertussis-infected human donors were screened for antibody reactivity with Bordetella iron-repressible cell envelope proteins. Pertussis patient sera recognized multiple iron-repressible proteins including the known outer membrane receptors for alcaligin, enterobactin and haem, supporting the hypothesis that B. pertussis is iron-starved and responds to the presence of diverse iron sources during natural infection.

Norepinephrine mediates acquisition of transferrin-iron in Bordetella bronchiseptica.

Previous research demonstrated that the sympathoadrenal catecholamine norepinephrine could promote the growth of Bordetella bronchiseptica in iron-restricted medium containing serum. In this study, norepinephrine was demonstrated to stimulate growth of this organism in the presence of partially iron-saturated transferrin but not lactoferrin. Although norepinephrine is known to induce transcription of the Bordetella bfeA enterobactin catechol xenosiderophore receptor gene, neither a bfeA mutant nor a bfeR regulator mutant was defective in growth responsiveness to norepinephrine. However, growth of a tonB mutant strain was not enhanced by norepinephrine, indicating that the response to this catecholamine was the result of high-affinity outer membrane transport. The B. bronchiseptica genome encodes a total of 19 known and predicted iron transport receptor genes, none of which, when mutated individually, were found to confer a defect in norepinephrine-mediated growth stimulation in the presence of transferrin. Labeling experiments demonstrated a TonB-dependent increase in cell-associated iron levels when bacteria grown in the presence of (55)Fe-transferrin were exposed to norepinephrine. In addition, TonB was required for maximum levels of cell-associated norepinephrine. Together, these results demonstrate that norepinephrine facilitates B. bronchiseptica iron acquisition from the iron carrier protein transferrin and this process may represent a mechanism by which some bacterial pathogens obtain this essential nutrient in the host environment.

Bordetella iron transport and virulence.

Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica are pathogens with a complex iron starvation stress response important for adaptation to nutrient limitation and flux in the mammalian host environment. The iron starvation stress response is globally regulated by the Fur repressor using ferrous iron as the co-repressor. Expression of iron transport system genes of Bordetella is coordinated by priority regulation mechanisms that involve iron source sensing. Iron source sensing is mediated by distinct transcriptional activators that are responsive to the cognate iron source acting as the inducer.

The Bordetella bfe system: growth and transcriptional response to siderophores, catechols, and neuroendocrine catecholamines.

Ferric enterobactin utilization by Bordetella bronchiseptica and Bordetella pertussis requires the BfeA outer membrane receptor. Under iron-depleted growth conditions, transcription of bfeA is activated by the BfeR regulator by a mechanism requiring the siderophore enterobactin. In this study, enterobactin-inducible bfeA transcription was shown to be TonB independent. To determine whether other siderophores or nonsiderophore catechols could be utilized by the Bfe system, various compounds were tested for the abilities to promote the growth of iron-starved B. bronchiseptica and induce bfeA transcription. The BfeA receptor transported ferric salmochelin, corynebactin, and the synthetic siderophores TRENCAM and MECAM. Salmochelin and MECAM induced bfeA transcription in iron-starved Bordetella cells, but induction by corynebactin and TRENCAM was minimal. The neuroendocrine catecholamines epinephrine, norepinephrine, and dopamine exhibited a remarkable capacity to induce transcription of bfeA. Norepinephrine treatment of B. bronchiseptica resulted in BfeR-dependent bfeA transcription, elevated BfeA receptor production, and growth stimulation. Pyrocatechol, carbidopa, and isoproterenol were similarly strong inducers of bfeA transcription, whereas tyramine and 3,4-dihydroxymandelic acid demonstrated low inducing activity. The results indicate that the inducer structure requires a catechol group for function and that the ability to induce bfeA transcription does not necessarily correlate with the ability to stimulate bacterial growth. The expanded range of catechol siderophores transported by the BfeA receptor demonstrates the potential versatility of the Bordetella Bfe iron retrieval system. The finding that catecholamine neurotransmitters activate bfeA transcription and promote growth suggests that Bordetella cells can perceive and may benefit from neuroendocrine catecholamines on the respiratory epithelium.