Toward trustworthy COVID-19 interventions: Building vaccine trust through community-university partnerships.

Prior research identifies trust as critical to increase vaccine acceptance and uptake. However, few intervention studies have sought to develop or test strategies for bolstering vaccine-related trust. To address this gap, this exploratory study identifies features of COVID-19 vaccine hesitancy interventions that can promote or undermine trust across three interconnected domains: institutional, interpersonal, and product (the vaccine itself). We draw on focus groups (N = 27 participants) with community and university partners involved with hosting COVID-19 testing and vaccine events in underserved Oklahoma communities. Focus groups explored participants' experiences serving community health needs and elicited feedback on proposed vaccine hesitancy interventions. Proposed interventions included two technology-based strategies (text message reminders and tablet-based testimonials and education) and one dialogue-based strategy (anti-body test interpretation). We find that community partners perceived local universities as trustworthy institutions because of their association with popular sports programs, academic credentials, and proximity, creating opportunities to address vaccine-related distrust through community-university partnerships. The most promising intervention strategies for building interpersonal trust included engaging in one-on-one dialogue and using autonomy enhancing approaches. Finally, interventions that successfully encouraged vaccine trust did so by incorporating personalized health information about individuals' potential level of protection and susceptibility to the COVID-19 virus. These findings can inform future public health efforts to create trustworthy vaccine hesitancy interventions.

CATCH-UP vaccines: protocol for a randomized controlled trial using the multiphase optimization strategy (MOST) framework to evaluate education interventions to increase COVID-19 vaccine uptake in Oklahoma.

BACKGROUND: Oklahoma's cumulative COVID-19 incidence is higher in rural than urban counties and higher than the overall US incidence. Furthermore, fewer Oklahomans have received at least one COVID-19 vaccine compared to the US average. Our goal is to conduct a randomized controlled trial using the multiphase optimization strategy (MOST) to test multiple educational interventions to improve uptake of COVID-19 vaccination among underserved populations in Oklahoma. METHODS: Our study uses the preparation and optimization phases of the MOST framework. We conduct focus groups among community partners and community members previously involved in hosting COVID-19 testing events to inform intervention design (preparation). In a randomized clinical trial, we test three interventions to improve vaccination uptake: (1) process improvement (text messages); (2) barrier elicitation and reduction (electronic survey with tailored questions/prompts); and (2) teachable moment messaging (motivational interviewing) in a three-factor fully crossed factorial design (optimization). DISCUSSION: Because of Oklahoma's higher COVID-19 impact and lower vaccine uptake, identifying community-driven interventions is critical to address vaccine hesitancy. The MOST framework provides an innovative and timely opportunity to efficiently evaluate multiple educational interventions in a single study. TRIAL REGISTRATION: ClinicalTrials.gov: NCT05236270, First Posted: February 11, 2022, Last Update Posted: August 31, 2022.

Antecedent Metabolic Health and Metformin (ANTHEM) Aging Study: Rationale and Study Design for a Randomized Controlled Trial.

The antidiabetic medication metformin has been proposed to be the first drug tested to target aging and extend healthspan in humans. While there is extensive epidemiological support for the health benefits of metformin in patient populations, it is not clear if these protective effects apply to those free of age-related disease. Our previous data in older adults without diabetes suggest a dichotomous change in insulin sensitivity and skeletal muscle mitochondrial adaptations after metformin treatment when co-prescribed with exercise. Those who entered the study as insulin-sensitive had no change to detrimental effects while those who were insulin-resistant had positive changes. The objective of this clinical trial is to determine if (a) antecedent metabolic health and (b) skeletal muscle mitochondrial remodeling and function mediate the positive or detrimental effects of metformin monotherapy, independent of exercise, on the metabolism and biology of aging. In a randomized, double-blind clinical trial, adults free of chronic disease (n = 148, 40-75 years old) are stratified as either insulin-sensitive or resistant based on homeostatic model assessment of insulin resistance (≤2.2 or ≥2.5) and take 1 500 mg/day of metformin or placebo for 12 weeks. Hyperinsulinemic-euglycemic clamps and skeletal muscle biopsies are performed before and after 12 weeks to assess primary outcomes of peripheral insulin sensitivity and mitochondrial remodeling and function. Findings from this trial will identify clinical characteristics and cellular mechanisms involved in modulating the effectiveness of metformin treatment to target aging that could inform larger Phase 3 clinical trials aimed at testing aging as a treatment indication for metformin. Clinical Trials Registration Number: NCT04264897.

Best Practices for Conducting Clinical Trials With Indigenous Children in the United States.

We provide guidance for conducting clinical trials with Indigenous children in the United States. We drew on extant literature and our experience to describe 3 best practices for the ethical and effective conduct of clinical trials with Indigenous children. Case examples of pediatric research conducted with American Indian, Alaska Native, and Native Hawaiian communities are provided to illustrate these practices. Ethical and effective clinical trials with Indigenous children require early and sustained community engagement, building capacity for Indigenous research, and supporting community oversight and ownership of research. Effective engagement requires equity, trust, shared interests, and mutual benefit among partners over time. Capacity building should prioritize developing Indigenous researchers. Supporting community oversight and ownership of research means that investigators should plan for data-sharing agreements, return or destruction of data, and multiple regulatory approvals. Indigenous children must be included in clinical trials to reduce health disparities and improve health outcomes in these pediatric populations. Establishment of the Environmental Influences on Child Health Outcomes Institutional Development Award States Pediatric Clinical Trials Network (ECHO ISPCTN) in 2016 creates a unique and timely opportunity to increase Indigenous children's participation in state-of-the-art clinical trials.

The modA10 phasevarion of nontypeable Haemophilus influenzae R2866 regulates multiple virulence-associated traits.

Non-typeable Haemophilus influenzae (NTHi) is a human restricted commensal and pathogen that elicits inflammation by adhering to and invading airway epithelia cells: transcytosis across these cells can result in systemic infection. NTHi strain R2866 was isolated from the blood of a normal 30-month old infant with meningitis, and is unusual for NTHi in that it is able to cause systemic infection. Strain R2866 is able to replicate in normal human serum due to expression of lgtC which mimics human blood group p(k). R2866 contains a phase-variable DNA methyltransferase, modA10 which switches ON and OFF randomly and reversibly due to polymerase slippage over a long tetrameric repeat tract located in its open reading frame. Random gain or loss of repeats during replication can results in expressed (ON), or not expressed (OFF) states, the latter due to a frameshift or transcriptional termination at a premature stop codon. We sought to determine if the unusual virulence of R2866 was modified by modA10 phase-variation. A modA10 knockout mutant was found to have increased adherence to, and invasion of, human ear and airway monolayers in culture, and increased invasion and transcytosis of polarized human bronchial epithelial cells. Intriguingly, the rate of bacteremia was lower in the infant rat model of infection than a wild-type R2866 strain, but the fatality rate was greater. Transcriptional analysis comparing the modA10 knockout to the R2866 wild-type parent strain showed increased expression of genes in the modA10 knockout whose products mediate cellular adherence. We conclude that loss of ModA10 function in strain R2866 enhances colonization and invasion by increasing expression of genes that allow for increased adherence, which can contribute to the increased virulence of this strain.

Draft Genome Sequences of Eight Nontypeable Haemophilus influenzae Strains Previously Characterized Using an Electrophoretic Typing Scheme.

Nontypeable Haemophilus influenzae is an important cause of human disease. Strains were selected for genome sequencing to represent the breadth of nontypeable strains within the species, as previously defined by the electrophoretic mobility of 16 metabolic enzymes.

Draft Genome Sequences of Six Nontypeable Haemophilus influenzae Strains That Establish Bacteremia in the Infant Rat Model of Invasive Disease.

Haemophilus influenzae is an important cause of invasive disease. The infant rat is the accepted model of invasive H. influenzae disease. Here, we report the genome sequences of six nontypeable H. influenzae strains that establish bacteremia in the infant rat.

Comparison of transcription of the Haemophilus influenzae iron/heme modulon genes in vitro and in vivo in the chinchilla middle ear.

BACKGROUND: Haemophilus influenzae is a significant cause of childhood otitis media, and also has an absolute growth requirement for heme. Recent microarray studies using three H. influenzae isolates were used to propose a putative core of genes responsive to iron and heme levels. Included in the core modulon were thirty seven genes that are preferentially expressed under iron/heme limitation, most of which are directly involved with iron and or heme acquisition. In this report, the core iron/heme modulon was further refined following microarray analysis of two additional nontypeable H. influenzae isolates from patients with otitis media. The transcriptional status of the genes comprising the refined iron/heme core modulon was then assessed in vivo, in a chinchilla model of otitis media. These in vivo experiments were performed to address the hypothesis that iron and heme regulated genes are both highly expressed in vivo and important, during clinical infection. RESULTS: Microarray analysis of two additional H. influenzae strains resulted in the definition of a core of iron/heme responsive genes. This core consisted of 35 genes maximally expressed under heme restriction and a further 20 genes maximally expressed in heme replete conditions. In vivo studies were performed with two nontypeable H. influenzae strains, 86-028NP and HI1722. The majority of operons identified as members of the core modulon by microarray were also actively upregulated in the chinchilla ear during otitis media. In 86-028NP, 70% of the operons were significantly upregulated while in HI1722 100% of the operons were upregulated in samples recovered from the chinchilla middle ear. CONCLUSION: This study elucidates a conserved core of H. influenzae genes the transcription of which is altered by the availability of iron and heme in the growth environment, and further assesses transcription of these genes in vivo. Elucidation of this modulon allows for identification of genes with unrecognized roles in iron/heme acquisition or homeostasis and/or potential roles in virulence. Defining these core genes is also of potential importance in identifying targets for therapeutic and vaccine designs since products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.

Haemophilus influenzae OxyR: characterization of its regulation, regulon and role in fitness.

To prevent damage by reactive oxygen species, many bacteria have evolved rapid detection and response systems, including the OxyR regulon. The OxyR system detects reactive oxygen and coordinates the expression of numerous defensive antioxidants. In many bacterial species the coordinated OxyR-regulated response is crucial for in vivo survival. Regulation of the OxyR regulon of Haemophilus influenzae was examined in vitro, and significant variation in the regulated genes of the OxyR regulon among strains of H. influenzae was observed. Quantitative PCR studies demonstrated a role for the OxyR-regulated peroxiredoxin/glutaredoxin as a mediator of the OxyR response, and also indicated OxyR self-regulation through a negative feedback loop. Analysis of transcript levels in H. influenzae samples derived from an animal model of otitis media demonstrated that the members of the OxyR regulon were actively upregulated within the chinchilla middle ear. H. influenzae mutants lacking the oxyR gene exhibited increased sensitivity to challenge with various peroxides. The impact of mutations in oxyR was assessed in various animal models of H. influenzae disease. In paired comparisons with the corresponding wild-type strains, the oxyR mutants were unaffected in both the chinchilla model of otitis media and an infant model of bacteremia. However, in weanling rats the oxyR mutant was significantly impaired compared to the wild-type strain. In contrast, in all three animal models when infected with a mixture of equal numbers of both wild-type and mutant strains the mutant strain was significantly out competed by the wild-type strain. These findings clearly establish a crucial role for OxyR in bacterial fitness.

Signature-tagging of a bacterial isolate demonstrates phenotypic variability of the progeny in vivo in the absence of defined mutations.

Awareness of the high degree of redundancy that occurs in several nutrient uptake pathways of Haemophilus influenzae led us to attempt to develop a quantitative STM method that could identify both null mutants and mutants with decreased fitness that remain viable in vivo. To accomplish this task we designed a modified STM approach that utilized a set of signature tagged wild-type (STWT) strains (in a single genetic background) as carriers for mutations in genes of interest located elsewhere in the genome. Each STWT strain differed from the others by insertion of a unique, Q-PCR-detectable, seven base pair tag into the same redundant gene locus. Initially ten STWTs were created and characterized in vitro and in vivo. As anticipated, the STWT strains were not significantly different in their in vitro growth. However, in the chinchilla model of otitis media, certain STWTs outgrew others by several orders of magnitude in mixed infections. Removal of the predominant STWT resulted in its replacement by a different predominant STWT on retesting. Unexpectedly we observed that the STWT exhibiting the greatest proliferation was animal dependent. These findings identify an inherent inability of the signature tag methodologies to accurately elucidate fitness in this animal model of infection and underscore the subtleties of H. influenzae gene regulation.

Identification of a siderophore utilization locus in nontypeable Haemophilus influenzae.

BACKGROUND: Haemophilus influenzae has an absolute aerobic growth requirement for either heme, or iron in the presence of protoporphyrin IX. Both iron and heme in the mammalian host are strictly limited in their availability to invading microorganisms. Many bacterial species overcome iron limitation in their environment by the synthesis and secretion of small iron binding molecules termed siderophores, which bind iron and deliver it into the bacterial cell via specific siderophore receptor proteins on the bacterial cell surface. There are currently no reports of siderophore production or utilization by H. influenzae. RESULTS: Comparative genomics revealed a putative four gene operon in the recently sequenced nontypeable H. influenzae strain R2846 that encodes predicted proteins exhibiting significant identity at the amino acid level to proteins involved in the utilization of the siderophore ferrichrome in other bacterial species. No siderophore biosynthesis genes were identified in the R2846 genome. Both comparative genomics and a PCR based analysis identified several additional H. influenzae strains possessing this operon. In growth curve assays strains containing the genes were able to utilize ferrichrome as an iron source. H. influenzae strains lacking the operon were unable to obtain iron from ferrichrome. An insertional mutation in one gene of the operon abrogated the ability of strains to utilize ferrichrome. In addition transcription of genes in the identified operon were repressible by high iron/heme levels in the growth media. CONCLUSIONS: We have identified an iron/heme-repressible siderophore utilization locus present in several nontypeable H. influenzae strains. The same strains do not possess genes encoding proteins associated with siderophore synthesis. The siderophore utilization locus may enable the utilization of siderophores produced by other microorganisms in the polymicrobial environmental niche of the human nasopharynx colonized by H. influenzae. This is the first report of siderophore utilization by H. influenzae.

Characterization of the Haemophilus influenzae tehB gene and its role in virulence.

The Haemophilus influenzae ORF designated HI1275 in the Rd KW20 genomic sequence encodes a putative S-adenosyl methyltransferase with significant similarity to tellurite-resistance determinants (tehB) in other species. While the H. influenzae tehB can complement an Escherichia coli tehB mutation, thus restoring tellurite resistance, its role in H. influenzae is unknown. In a previous study defining the iron and haem modulon of H. influenzae, we showed that transcription of this gene in H. influenzae Rd KW20 increases during growth in iron- and haem-restricted media. Since iron and haem uptake genes, and other known virulence factors, constitute the majority of the iron- and haem-regulated gene set, we postulated that tehB may play a role in nutrient acquisition and/or the virulence of H. influenzae. A tehB mutant was constructed in the H. influenzae type b strain 10810 and was evaluated for growth defects in various supplemented media, as well as for its ability to cause infection in rat models of infection. Deletion of tehB leads to an increase in sensitivity both to tellurite and to the oxidizing agents cumene hydroperoxide, tert-butyl hydroperoxide and hydrogen peroxide. The tehB mutant additionally showed a significantly reduced ability to utilize free haem as well as several haem-containing moieties including haem-human serum albumin, haemoglobin and haemoglobin-haptoglobin. Examination of the regulation kinetics indicated that transcription of tehB was independent of both tellurite exposure and oxidative stress. Paired comparisons of the tehB mutant and the wild-type H. influenzae strain 10810 showed that tehB is required for wild-type levels of infection in rat models of H. influenzae invasive disease. To our knowledge this is the first report of a role for tehB in virulence in any bacterial species. These data demonstrate that H. influenzae tehB plays a role in both resistance to oxidative damage and haem uptake/utilization, protects H. influenzae from tellurite exposure, and is important for virulence of this organism in an animal model of invasive disease.

The dppBCDF gene cluster of Haemophilus influenzae: Role in heme utilization.

BACKGROUND: Haemophilus influenzae requires a porphyrin source for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. This porphyrin requirement may be satisfied by either heme alone, or protoporphyrin IX in the presence of an iron source. One protein involved in heme acquisition by H. influenzae is the periplasmic heme binding protein HbpA. HbpA exhibits significant homology to the dipeptide and heme binding protein DppA of Escherichia coli. DppA is a component of the DppABCDF peptide-heme permease of E. coli. H. influenzae homologs of dppBCDF are located in the genome at a point distant from hbpA. The object of this study was to investigate the potential role of the H. influenzae dppBCDF locus in heme utilization. FINDINGS: An insertional mutation in dppC was constructed and the impact of the mutation on the utilization of both free heme and various proteinaceous heme sources as well as utilization of protoporphyrin IX was determined in growth curve studies. The dppC insertion mutant strain was significantly impacted in utilization of all tested heme sources and protoporphyin IX. Complementation of the dppC mutation with an intact dppCBDF gene cluster in trans corrected the growth defects seen in the dppC mutant strain. CONCLUSION: The dppCBDF gene cluster constitutes part of the periplasmic heme-acquisition systems of H. influenzae.

The heme-binding protein (HbpA) of Haemophilus influenzae as a virulence determinant.

Haemophilus influenzae has an absolute growth requirement for heme and the heme-binding lipoprotein (HbpA) and has been implicated in the utilization of this essential nutrient. We constructed an insertional mutation of hbpA in a type b and a nontypeable H. influenzae strain. In the type b strain, the hbpA mutant was impaired in utilization of heme complexed to either hemopexin or to albumin and in the utilization of low levels of heme but not in the utilization of heme at high levels or of hemoglobin or hemoglobin-haptoglobin complexes. In contrast, the hbpA mutant derivative of the nontypeable strain was impaired in utilization of all tested heme sources. We further examined the impact of the hbpA mutation in animal models of H. influenzae disease. The hbpA mutant of the nontypeable strain was indistinguishable from the wild-type strain in the chinchilla model of otitis media. The hbpA mutant derivative of the type b strain caused bacteremia as well as the wild-type strain in 5-day old infant rats. However, in 30-day old rats the hbpA caused significantly lower rates of bacteremia than the wild-type strain indicating a role for hbpA and heme acquisition in virulence in this model of H. influenzae disease. In conclusion, HbpA is important for heme utilization by multiple H. influenzae strains and is a virulence determinant in a model of H. influenzae invasive disease.

The iron/heme regulated genes of Haemophilus influenzae: comparative transcriptional profiling as a tool to define the species core modulon.

BACKGROUND: Haemophilus influenzae requires heme for aerobic growth and possesses multiple mechanisms to obtain this essential nutrient. Although an understanding of the heme acquisition mechanisms of H. influenzae is emerging, significant gaps in our knowledge remain. Unresolved issues include the identities of all genes exhibiting altered transcription in response to iron and heme availability, the fraction of such genes functioning in iron/heme acquisition, and the heterogeneity of this gene set among clinical isolates. Previously we utilized H. influenzae strain Rd KW20 to demonstrate the utility of transcriptional profiling in defining the genes exhibiting altered transcription in response to environmental iron and heme levels. The current study expands upon those observations by determining the iron/heme modulons of two clinical isolates, the type b isolate 10810 and the nontypeable isolate R2866. These data are used to begin to define the core iron/heme modulon of the species. RESULTS: Microarray studies were performed to compare gene expression on transition from iron/heme-restricted to iron/heme-replete conditions for each isolate. Of 1820 ORFs on the array corresponding to R2866 genes, 363 were significantly differentially expressed: 233 were maximally transcribed under iron/heme-replete conditions and 130 under iron/heme-restricted conditions. Of the 1883 ORFs representing genes of strain 10810, 353 were significantly differentially transcribed: 150 were preferentially transcribed under iron/heme-replete conditions and 203 under iron/heme-restricted conditions. Comparison of the data sets indicated that 163 genes exhibited similar regulation in both isolates and that 74 of these exhibited similar patterns of regulation in Rd KW20. These comprise the putative core iron/heme modulon. CONCLUSION: This study provides evidence for a conserved core of H. influenzae genes the transcription of which is altered by the availability of iron and/or heme in the growth environment. Elucidation of this modulon provides a means to identify genes with unrecognized roles in iron/heme acquisition or homeostasis, unanticipated responsiveness to environmental levels of the micronutrients or potential roles in virulence. Defining these core genes is also of potential importance in identifying targets for therapeutic and vaccine designs since products of these genes are likely to be preferentially expressed during growth in iron/heme restricted sites of the human body.

Catalase as a source of both X- and V-factor for Haemophilus influenzae.

Haemophilus influenzae requires two growth factors, designated factor X (porphyrin) and factor V (NAD). Mammalian catalases contain both bound heme and NADPH. This study shows that catalase can supply both factors X and V to H. influenzae in vitro, thus representing a potential in vivo source of these essential growth factors.

Lipoprotein e (P4) of Haemophilus influenzae: role in heme utilization and pathogenesis.

Lipoprotein e (P4) of Haemophilus influenzae is a phosphomonoesterase, encoded by the hel gene, that has been implicated in the acquisition of heme by this fastidious organism. However, lipoprotein e (P4) is also involved in the utilization of NAD and NMN. Some reports have concluded that the reported heme-related growth defect actually reflects a growth defect for NAD. In the current study, hel insertion mutants were constructed and a role for e (P4) in heme acquisition was demonstrated independent of its role in NAD or NMN acquisition. In addition, a rat model of infection demonstrated a role for e (P4) in the pathogenesis of invasive disease.

The haem-haemopexin utilization gene cluster (hxuCBA) as a virulence factor of Haemophilus influenzae.

Haemophilus influenzae has an absolute growth requirement for a porphyrin source, which can be supplied in vitro by haem, haemoglobin, or the haemoglobin-haptoglobin, haem-haemopexin and haem-albumin complexes. Utilization of the haem-haemopexin complex is known to be mediated by the products of the hxuCBA gene cluster. It was demonstrated that hxuC, but not hxuA or hxuB, is also essential for the utilization of haem from haem-albumin complexes. Mutants of the type b strain E1a lacking genes in the hxuCBA gene cluster were examined for their ability to cause bacteraemia in rat models of invasive disease. In 5-day-old rats, mutants in the hxuCBA genes yielded a significantly reduced bacteraemic titre compared to the wild-type strain. In addition, 5-day-old rats infected with the hxuCBA mutant strains exhibited significantly improved survival rates compared to those infected with the wild-type strain. Mutations in the haemoglobin/haemoglobin-haptoglobin-binding protein genes (hgps), either alone or in combination with the hxuCBA mutations, had no impact on virulence in 5-day-old rats. In 30-day-old rats infected with either the hxuCBA mutants or the wild-type strains, there was no significant difference in the ability to establish bacteraemia although bacterial titres were lower in rats infected with the hxuCBA mutants than in those infected with the wild-type strain. These age-related differences in the impact of mutations in the hxuCBA gene cluster may be related to changes in levels of host haem-binding proteins during development of the rat.

Complex role of hemoglobin and hemoglobin-haptoglobin binding proteins in Haemophilus influenzae virulence in the infant rat model of invasive infection.

Haemophilus influenzae requires an exogenous heme source for aerobic growth in vitro. Hemoglobin or hemoglobin-haptoglobin satisfies this requirement. Heme acquisition from hemoglobin-haptoglobin is mediated by proteins encoded by hgp genes. Both Hgps and additional proteins, including those encoded by the hxu operon, provide independent pathways for hemoglobin utilization. Recently we showed that deletion of the set of three hgp genes from a nontypeable strain (86-028NP) of H. influenzae attenuated virulence in the chinchilla otitis media model of noninvasive disease. The present study was undertaken to investigate the role of the hgp genes in virulence of the wild-type serotype b clinical isolate HI689 in the infant rat model of hematogenous meningitis, an established model of invasive disease requiring aerobic growth. Bacteremia of high titer and long duration (>14 days) and histopathologically confirmed meningitis occurred in >95% of infant rats challenged at 5 days of age with strain HI689. While mutations disrupting either the Hgp- or Hxu-mediated pathway of heme acquisition had no effect on virulence in infant rats, an isogenic mutant deficient for both pathways was unable to sustain bacteremia or produce meningitis. In contrast, mutations disrupting either pathway decreased the limited ability of H. influenzae to initiate and sustain bacteremia in weanling rats. Biochemical and growth studies also indicated that infant rat plasma contains multiple heme sources that change with age. Taken together, these data indicate that both the hgp genes and the hxuC gene are virulence determinants in the rat model of human invasive disease.

Transcriptional profile of Haemophilus influenzae: effects of iron and heme.

Haemophilus influenzae requires either heme or a porphyrin and iron source for growth. Microarray studies of H. influenzae strain Rd KW20 identified 162 iron/heme-regulated genes, representing approximately 10% of the genome, with > or =1.5-fold changes in transcription in response to iron/heme availability in vitro. Eighty genes were preferentially expressed under iron/heme restriction; 82 genes were preferentially expressed under iron/heme-replete conditions.