Epidemiologic, Immunologic, and Virus Characteristics in Patients With Paired Severe Acute Respiratory Syndrome Coronavirus 2 Serology and Reverse-Transcription Polymerase Chain Reaction Testing.

BACKGROUND: The natural history and clinical progression of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections can be better understood using combined serological and reverse-transcription polymerase chain reaction (RT-PCR) testing. METHODS: Nasopharyngeal swabs and serum were collected at a single time-point from patients at an urban, public hospital during August-November 2020 and tested for SARS-CoV-2 using RT-PCR, viral culture, and anti-spike pan-immunoglobulin antibody testing. Participant demographics and symptoms were collected through interview. The χ 2 and Fisher exact tests were used to identify associations between RT-PCR and serology results with presence of viable virus and frequency of symptoms. RESULTS: Among 592 participants, 129 (21.8%) had evidence of SARS-CoV-2 infection by RT-PCR or serology. Presence of SARS-CoV-2 antibodies was strongly associated with lack of viable virus (P = .016). COVID-19 symptom frequency was similar for patients testing RT-PCR positive/seronegative and patients testing RT-PCR positive/seropositive. Patients testing RT-PCR positive/seronegative reported headaches, fatigue, diarrhea, and vomiting at rates not statistically significantly different from those testing RT-PCR negative/seropositive. CONCLUSIONS: While patients testing SARS-CoV-2 seropositive were unlikely to test positive for viable virus and were therefore at low risk for forward transmission, coronavirus disease 2019 (COVID-19) symptoms were common. Paired SARS-CoV-2 RT-PCR and antibody testing provides more nuanced understanding of patients' COVID-19 status.

SARS-CoV-2 Infection and Mitigation Efforts among Office Workers, Washington, DC, USA.

Despite mitigation efforts, 2 coronavirus disease outbreaks were identified among office workers in Washington, DC. Moderate adherence to workplace mitigation efforts was reported in a serologic survey; activities outside of the workplace were associated with infection. Adherence to safety measures are critical for returning to work during the pandemic.

Effects of Patient Characteristics on Diagnostic Performance of Self-Collected Samples for SARS-CoV-2 Testing.

We evaluated the performance of self-collected anterior nasal swab (ANS) and saliva samples compared with healthcare worker-collected nasopharyngeal swab specimens used to test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We used the same PCR diagnostic panel to test all self-collected and healthcare worker-collected samples from participants at a public hospital in Atlanta, Georgia, USA. Among 1,076 participants, 51.9% were men, 57.1% were >50 years of age, 81.2% were Black (non-Hispanic), and 74.9% reported >1 chronic medical condition. In total, 8.0% tested positive for SARS-CoV-2. Compared with nasopharyngeal swab samples, ANS samples had a sensitivity of 59% and saliva samples a sensitivity of 68%. Among participants tested 3-7 days after symptom onset, ANS samples had a sensitivity of 80% and saliva samples a sensitivity of 85%. Sensitivity varied by specimen type and patient characteristics. These findings can help physicians interpret PCR results for SARS-CoV-2.

Q Fever: A troubling disease and a challenging diagnosis.

Q fever is a disease caused by the bacterial pathogen Coxiella burnetii. This hardy organism can easily spread long distances in the wind, and only a few infectious aerosolized particles are necessary to cause serious illness. Presentations of Q fever disease can be wide-ranging, allowing it to masquerade as other illnesses and highlight the importance of laboratory testing for diagnosis and treatment. This review summarizes Q fever's epidemiology and clinical presentations and presents classical laboratory diagnostic assays and novel approaches to detecting this troubling disease.

IscR is essential for yersinia pseudotuberculosis type III secretion and virulence.

Type III secretion systems (T3SS) are essential for virulence in dozens of pathogens, but are not required for growth outside the host. Therefore, the T3SS of many bacterial species are under tight regulatory control. To increase our understanding of the molecular mechanisms behind T3SS regulation, we performed a transposon screen to identify genes important for T3SS function in the food-borne pathogen Yersinia pseudotuberculosis. We identified two unique transposon insertions in YPTB2860, a gene that displays 79% identity with the E. coli iron-sulfur cluster regulator, IscR. A Y. pseudotuberculosis iscR in-frame deletion mutant (ΔiscR) was deficient in secretion of Ysc T3SS effector proteins and in targeting macrophages through the T3SS. To determine the mechanism behind IscR control of the Ysc T3SS, we carried out transcriptome and bioinformatic analysis to identify Y. pseudotuberculosis genes regulated by IscR. We discovered a putative IscR binding motif upstream of the Y. pseudotuberculosis yscW-lcrF operon. As LcrF controls transcription of a number of critical T3SS genes in Yersinia, we hypothesized that Yersinia IscR may control the Ysc T3SS through LcrF. Indeed, purified IscR bound to the identified yscW-lcrF promoter motif and mRNA levels of lcrF and 24 other T3SS genes were reduced in Y. pseudotuberculosis in the absence of IscR. Importantly, mice orally infected with the Y. pseudotuberculosis ΔiscR mutant displayed decreased bacterial burden in Peyer's patches, mesenteric lymph nodes, spleens, and livers, indicating an essential role for IscR in Y. pseudotuberculosis virulence. This study presents the first characterization of Yersinia IscR and provides evidence that IscR is critical for virulence and type III secretion through direct regulation of the T3SS master regulator, LcrF.

The membrane protein PrsS mimics σS in protecting Staphylococcus aureus against cell wall-targeting antibiotics and DNA-damaging agents.

Staphylococcus aureus possesses a lone extracytoplasmic function (ECF) sigma factor, σ(S). In Bacillus subtilis, the ECF sigma factor, σ(W), is activated through a proteolytic cascade that begins with cleavage of the RsiW anti-sigma factor by a site-1 protease (S1P), PrsW. We have identified a PrsW homologue in S. aureus (termed PrsS) and explored its role in σ(S) regulation. Herein, we demonstrate that although a cognate σ(S) anti-sigma factor currently remains elusive, prsS phenocopies sigS in a wealth of regards. Specifically, prsS expression mimics the upregulation observed for sigS in response to DNA-damaging agents, cell wall-targeting antibiotics and during ex vivo growth in human serum and murine macrophages. prsS mutants also display the same sensitivities of sigS mutants to the DNA-damaging agents methyl methane sulfonate (MMS) and hydrogen peroxide, and the cell wall-targeting antibiotics ampicillin, bacitracin and penicillin-G. These phenotypes appear to be explained by alterations in abundance of proteins involved in drug resistance (Pbp2a, FemB, HmrA) and the response to DNA damage (BmrA, Hpt, Tag). Our findings seem to be mediated by putative proteolytic activity of PrsS, as site-directed mutagenesis of predicted catalytic residues fails to rescue the sensitivity of the mutant to H2O2 and MMS. Finally, a role for PrsS in S. aureus virulence was identified using human and murine models of infection. Collectively, our data indicate that PrsS and σ(S) function in a similar manner, and perhaps mediate virulence and resistance to DNA damage and cell wall-targeting antibiotics, via a common pathway.

Investigating the genetic regulation of the ECF sigma factor σS in Staphylococcus aureus.

BACKGROUND: We previously identified an ECF sigma factor, σS, that is important in the stress and virulence response of Staphylococcus aureus. Transcriptional profiling of sigS revealed that it is differentially expressed in many laboratory and clinical isolates, suggesting the existence of regulatory networks that modulates its expression. RESULTS: To identify regulators of sigS, we performed a pull down assay using S. aureus lysates and the sigS promoter. Through this we identified CymR as a negative effector of sigS expression. Electrophoretic mobility shift assays (EMSAs) revealed that CymR directly binds to the sigS promoter and negatively effects transcription. To more globally explore genetic regulation of sigS, a Tn551 transposon screen was performed, and identified insertions in genes that are involved in amino acid biosynthesis, DNA replication, recombination and repair pathways, and transcriptional regulators. In efforts to identify gain of function mutations, methyl nitro-nitrosoguanidine mutagenesis was performed on a sigS-lacZ reporter fusion strain. From this a number of clones displaying sigS upregulation were subject to whole genome sequencing, leading to the identification of the lactose phosphotransferase repressor, lacR, and the membrane histidine kinase, kdpD, as central regulators of sigS expression. Again using EMSAs we determined that LacR is an indirect regulator of sigS expression, while the response regulator, KdpE, directly binds to the promoter region of sigS. CONCLUSIONS: Collectively, our work suggests a complex regulatory network exists in S. aureus that modulates expression of the ECF sigma factor, σS.

Efficacy of rifapentine and other rifamycins against Coxiella burnetii in vitro.

Since 1999, doxycycline and hydroxychloroquine have been the recommended treatment for chronic Q fever, a life-threatening disease caused by the bacterial pathogen, Coxiella burnetii. Despite the duration of its use, the treatment is not ideal due to the lengthy treatment time, high mortality rate, resistant strains, and the potential for contraindicated usage. A literature search was conducted to identify studies that screened large panels of drugs against C. burnetii to identify novel targets with potential efficacy against C. burnetii. Twelve candidate antimicrobials approved for use in humans by the US Food and Drug Administration were selected and minimum inhibitory concentrations (MICs) were determined against the low virulence strain Nine Mile phase II. Rifabutin and rifaximin were the best performing antibiotics tested with MICs of ≤0.01 µg mL-1. Further screening of these top candidates was conducted alongside two drugs from the same class, rifampin, well-characterized, and rifapentine, not previously reported against C. burnetii. These were screened against virulent strains of C. burnetii representing three clinically relevant genotypes. Rifapentine was the most effective in the human monocytic leukemia cell line, THP-1, with a MIC ≤0.01 µg mL-1. In the human kidney epithelial cell line, A-498, efficacy of rifapentine, rifampin, and rifabutin varied across C. burnetii strains with MICs between ≤0.001 and 0.01 µg mL-1. Rifampin, rifabutin, and rifapentine were all bactericidal against C. burnetii; however, rifabutin and rifapentine demonstrated impressive bactericidal activity as low as 0.1 µg mL-1 and should be further explored as alternative Q fever treatments given their efficacy in vitro. IMPORTANCE: This work will help inform investigators and physicians about potential alternative antimicrobial therapies targeting the causative agent of Q fever, Coxiella burnetii. Chronic Q fever is difficult to treat, and alternative antimicrobials are needed. This manuscript explores the efficacy of rifamycin antibiotics against virulent strains of C. burnetii representing three clinically relevant genotypes in vitro. Importantly, this study determines the susceptibility of C. burnetii to rifapentine, which has not been previously reported. Evaluation of the bactericidal activity of the rifamycins reveals that rifabutin and rifapentine are bactericidal at low concentrations, which is unusual for antibiotics against C. burnetii.

Longitudinal surveillance of Coxiella burnetii following an abortion storm in domestic goats.

Q fever is a disease caused by Coxiella burnetii, which can cause serious illness in humans and abortions in goats. A Q fever outbreak among an unvaccinated goat herd led to a 65% loss of the kid crop in spring 2018. To assess the impact of the outbreak on the herd and environment, longitudinal surveillance of the ranch was conducted across three samplings in September 2018, April 2019, and May 2022. Antibodies against C. burnetii were monitored by an indirect immunofluorescence assay. Shedding was monitored through analysis of vaginal/fecal swabs and milk. Environmental swabs and bulk soil were collected from various locations around the ranch. Animal and environmental samples were analyzed for C. burnetii DNA by PCR. Herd-level seroprevalence decreased from 89% in 2018 to 84.3% in 2019, and 64.5% in 2022. Overall herd shedding was 14.4% in 2018, 7.4% in 2019, and 6.7% in 2022. The percentage of C. burnetii-positive environmental samples was 83.7% in 2018, 51.7% in 2019, and 28.6% in 2022. Serological evidence suggests that new infections were occurring in the herd 4 years post-abortion storm. This study demonstrates the presence of C. burnetii shedding and environmental contamination in a goat operation at least four kidding seasons after an outbreak. A better understanding of management practices that can improve outcomes for infected herds, particularly in areas without access to vaccines against C. burnetii, is needed to better protect operators and the public.

Coxiella burnetii in domestic doe goats in the United States, 2019-2020.

Coxiella burnetii is a bacterial pathogen capable of causing serious disease in humans and abortions in goats. Infected goats can shed C. burnetii through urine, feces, and parturient byproducts, which can lead to infections in humans when the bacteria are inhaled. Goats are important C. burnetii reservoirs as evidenced by goat-related outbreaks across the world. To better understand the current landscape of C. burnetii infection in the domestic goat population, 4,121 vaginal swabs from 388 operations across the United States were analyzed for the presence of C. burnetii by IS1111 PCR as part of the United States Department of Agriculture, Animal Plant Health Inspection Service, Veterinary Services' National Animal Health Monitoring System Goats 2019 Study. In total, 1.5% (61/4121) of swabs representing 10.3% (40/388) (weighted estimate of 7.8, 95% CI 4.4-13.5) of operations were positive for C. burnetii DNA. The quantity of C. burnetii on positive swabs was low with an average Ct of 37.9. Factors associated with greater odds of testing positive included suspected Q fever in the herd in the previous 3 years, the presence of wild deer or elk on the operation, and the utilization of hormones for estrus synchronization. Factors associated with reduced odds of testing positive include the presence of kittens and treatment of herds with high tannin concentrate plants, diatomaceous earth, and tetrahydropyrimidines. In vitro analysis demonstrated an inhibitory effect of the tetrahydropyrimidine, pyrantel pamoate, on the growth of C. burnetii in axenic media as low as 1 µg per mL. The final multivariable logistic regression modeling identified the presence of wild predators on the operation or adjacent property (OR = 9.0, 95% CI 1.3-61.6, p value = 0.0248) as a risk factor for C. burnetii infection.

The extracytoplasmic function sigma factor σS protects against both intracellular and extracytoplasmic stresses in Staphylococcus aureus.

Previously we identified a novel component of the Staphylococcus aureus regulatory network, an extracytoplasmic function σ-factor, σ(S), involved in stress response and disease causation. Here we present additional characterization of σ(S), demonstrating a role for it in protection against DNA damage, cell wall disruption, and interaction with components of the innate immune system. Promoter mapping reveals the existence of three unique sigS start sites, one of which appears to be subject to autoregulation. Transcriptional profiling revealed that sigS expression remains low in a number of S. aureus wild types but is upregulated in the highly mutated strain RN4220. Further analysis demonstrates that sigS expression is inducible upon exposure to a variety of chemical stressors that elicit DNA damage, including methyl methanesulfonate and ciprofloxacin, as well as those that disrupt cell wall stability, such as ampicillin and oxacillin. Significantly, expression of sigS is highly induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Phenotypically, σ(S) mutants display sensitivity to a broad range of DNA-damaging agents and cell wall-targeting antibiotics. Furthermore, the survivability of σ(S) mutants is strongly impacted during challenge by components of the innate immune system. Collectively, our data suggest that σ(S) likely serves dual functions within the S. aureus cell, protecting against both cytoplasmic and extracytoplasmic stresses. This further argues for its important, and perhaps novel, role in the S. aureus stress and virulence responses.

The impact of CodY on virulence determinant production in community-associated methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a leading human pathogen of both hospital and community-associated diseases worldwide. This organism causes a wealth of infections within the human host as a result of the vast arsenal of toxins encoded within its genome. Previous transcriptomic studies have shown that toxin production in S. aureus can be strongly impacted by the negative regulator CodY. CodY acts by directly, and indirectly (via Agr), repressing toxin production during times of plentiful nutrition. In this study, we use iTRAQ-based proteomics for the first time to study virulence determinant production in S. aureus, so as to correlate transcriptional observations with actual changes in protein synthesis. Using a codY mutant in the epidemic CA-MRSA clone USA300 we demonstrate that deletion of this transcription factor results in a major upregulation of toxin synthesis in both post-exponential and stationary growth. Specifically, we observe hyper-production of secreted proteases, leukocidins and hemolysins in both growth phases in the USA300 codY mutant. Our findings demonstrate the power of mass spectrometry-based quantitative proteomics for studying toxin production in S. aureus, and the importance of CodY to this central process in disease causation and infection.

NsaRS is a cell-envelope-stress-sensing two-component system of Staphylococcus aureus.

Staphylococcus aureus possesses 16 two-component systems (TCSs), two of which (GraRS and NsaRS) belong to the intramembrane-sensing histidine kinase (IM-HK) family, which is conserved within the firmicutes. NsaRS has recently been documented as being important for nisin resistance in S. aureus. In this study, we present a characterization of NsaRS and reveal that, as with other IM-HK TCSs, it responds to disruptions in the cell envelope. Analysis using a lacZ reporter-gene fusion demonstrated that nsaRS expression is upregulated by a variety of cell-envelope-damaging antibiotics, including phosphomycin, ampicillin, nisin, gramicidin, carbonyl cyanide m-chlorophenylhydrazone and penicillin G. Additionally, we reveal that NsaRS regulates a downstream transporter NsaAB during nisin-induced stress. NsaS mutants also display a 200-fold decreased ability to develop resistance to the cell-wall-targeting antibiotic bacitracin. Microarray analysis reveals that the transcription of 245 genes is altered in an nsaS mutant, with the vast majority being downregulated. Included within this list are genes involved in transport, drug resistance, cell envelope synthesis, transcriptional regulation, amino acid metabolism and virulence. Using inductively coupled plasma-MS we observed a decrease in intracellular divalent metal ions in an nsaS mutant when grown under low abundance conditions. Characterization of cells using electron microscopy reveals that nsaS mutants have alterations in cell envelope structure. Finally, a variety of virulence-related phenotypes are impaired in nsaS mutants, including biofilm formation, resistance to killing by human macrophages and survival in whole human blood. Thus, NsaRS is important in sensing cell damage in S. aureus and functions to reprogram gene expression to modify cell envelope architecture, facilitating adaptation and survival.

Comparison of three Coxiella burnetii infectious routes in mice.

Evidence suggests that Coxiella burnetii, which is shed in the milk, urine, feces, and birth products of infected domestic ruminants, can lead to Q fever disease following consumption of unpasteurized dairy products; however, C. burnetii is not believed to be a major gastrointestinal pathogen. Most infections are associated with inhalation of aerosols generated from the excreta of domestic ruminants. We recently demonstrated that C. burnetii delivered by oral gavage (OG) resulted in dissemination and an immune response; however, it is unclear how infection via the oral route compares to other well-established routes. Therefore, we delivered three strains of C. burnetii (representing three pertinent sequence types in the United States, such as ST16, ST20, and ST8) to immunocompetent mice in four doses via aerosol challenge (AC), intraperitoneal injection (IP), or OG. Low dose (10^5) of ST16 by OG was insufficient to cause infection, yet doses 1,000- or 100-fold lower by IP or AC, respectively, induced a robust immune response and dissemination. Despite being able to induce an immune response in a dose-dependent manner, administration of C. burnetii via OG is the least efficient route tested. Not only were the immune responses and bacterial loads diminished in mice exposed by OG relative to AC or IP, the efficiency of transmission was also inferior. High doses (10^8) were not sufficient to ensure transmission to 100% of the ST20 or ST8 cohorts. These results may provide some basis for why ingestion of C. burnetii as a mode of Q fever transmission is not often reported.

Coxiella burnetii infections in mice: Immunological responses to contemporary genotypes found in the US.

Coxiella burnetii is an obligate intracellular bacterium that causes the human disease Q fever, which can manifest as an acute flu-like illness or a long-term chronic illness, such as endocarditis. Three genotypes (ST8, ST16, and ST20) of Coxiella burnetii are commonly found in the contemporary US and are associated with specific animal hosts. Although all three genotypes have been isolated from humans with Q fever, studies comparing virulence between C. burnetii sequence types have been rare. Here, groups of mice were infected via aerosol inoculation with isolates derived from cow's milk, environmental, animal, and human samples. Mice were monitored for weight loss and blood samples were takenweekly. Animals were euthanized at 2- and 12-weeks post-infection, and bacterial burden was determined for tissues by real-time PCR. The levels of anti-Coxiella antibodies and selected inflammatory cytokines were determined for serum samples. Weight loss and splenomegaly were observed in mice infected with ST20 and ST16 isolates but were absent in the mice infected with ST8 isolates. Bacterial concentrations in the tissues were lower in the ST8 isolates at 2 weeks post-infection relative to all other isolates. ST16 and ST20 isolates induced robust antibody and cytokine responses, while ST8 isolates produced significantly lower anti-C. burnetii titers early in the infection but saw increased titers in some animals several weeks post-infection. The data suggest that the ST8 isolates are less virulent in this mouse model, as they produce less robust antibody responses that are slow to develop, relative to the ST16 and ST20 isolates.

Association Between Serological Responses to Two Zoonotic Ruminant Pathogens and Esophageal Squamous Cell Carcinoma.

Questionnaire data have linked contact with ruminants to the risk of esophageal squamous cell carcinoma (ESCC) in high-risk Asian populations. To better understand this observed association, we investigated exposure to two major zoonotic ruminant pathogens relative to ESCC risk. Using enzyme-linked immunosorbent assay, immunofluorescence assay, and Brucella microagglutination test assays, we measured immunoglobulin G anti-Coxiella burnetii and anti-Brucella spp. antibodies in patients with ESCC (n = 177) and population-based controls (n = 177) matched by age, gender, and residence area from the Golestan case-control study in Iran. We found a similarly high seroprevalence of C. burnetii in ESCC cases and controls (75% and 80%, respectively), and a similarly low seroprevalence of Brucella spp. (0% and 0.6%, respectively). While documenting a high exposure to one of two zoonotic ruminant infections, this exposure failed to explain the observed association of ruminant contact and ESCC risk in this high-risk population.

Analysis of recombinant proteins for Q fever diagnostics.

Serology is essential for Q fever diagnostics, a disease caused by the bacterial pathogen Coxiella burnetii. The gold standard test is an immunofluorescence assay utilizing whole cell antigens, which are both dangerous and laborious to produce. Complexities of the antigen coupled with the subjective nature of the assay lead to decreased uniformity of test results and underscore the need for improved methodologies. Thirty-three C. burnetii proteins, previously identified as immunoreactive, were screened for reactivity to naturally infected goat serum. Based on reactivity, 10 proteins were analyzed in a secondary screen against human serum from healthy donors. Assay sensitivity and specificity ranged from 21 to 71% and 90 to 100%, respectively. Three promising antigens were identified based on receiver operating characteristic curve analysis (CBU_1718, CBU_0307, and CBU_1398). Five multiplex assays failed to outperform the individual proteins, with sensitivities and specificities ranging from 29 to 57% and 90 to 100%, respectively. Truncating the top antigen, CBU_1718, had no effect on specificity (90%); yet sensitivity decreased dramatically (71% to 21%). Through this study, we have expanded the subset of C. burnetii immunoreactive proteins validated by enzyme-linked immunosorbent assay and demonstrate the effect of novel antigen combinations and protein truncations on assay performance.

Identification of a unique transcriptional architecture for the sigS operon in Staphylococcus aureus.

Staphylococcus aureus possess three alternative σ factors, including a lone extracytoplasmic function σ factor, σS. Our group previously identified and characterized this element, mapping three sigS promoters, demonstrating its inducibility during stress and virulence inducing conditions and demonstrating a role for this factor in disease causation. In the present study, we identify a fourth promoter of the sigS operon, termed P4, located in a unique position internal to the sigS coding region. Transcriptional profiling revealed that expression from P4 is dominant to the three upstream promoters, particularly upon exposure to chemical stressors that elicit DNA damage and disrupt cell wall stability; each of which have previously been shown to stimulate sigS expression. Importantly, expression of this fourth promoter, followed by at least one or more of the upstream promoters, is induced during growth in serum and upon phagocytosis by RAW 264.7 murine macrophage-like cells. Finally, we demonstrate that a downstream gene, SACOL1829, bears a large 3΄ UTR that spans the sigS-SACOL1828 coding region, and may serve to compete with the P4 transcript to inhibit σS production. Collectively, these findings reveal a unique operon architecture for the sigS locus that indicates the potential for novel regulatory mechanisms governing its expression.

Control of hmu Heme Uptake Genes in Yersinia pseudotuberculosis in Response to Iron Sources.

Despite the mammalian host actively sequestering iron to limit pathogenicity, heme (or hemin when oxidized) and hemoproteins serve as important sources of iron for many bloodborne pathogens. The HmuRSTUV hemin uptake system allows Yersinia species to uptake and utilize hemin and hemoproteins as iron sources. HmuR is a TonB-dependent outer membrane receptor for hemin and hemoproteins. HmuTUV comprise a inner membrane ABC transporter that transports hemin and hemoproteins from the periplasmic space into the bacterial cytoplasm, where it is degraded by HmuS. Here we show that hmuSTUV but not hmuR are expressed under iron replete conditions, whereas hmuR as well as hmuSTUV are expressed under iron limiting conditions, suggesting complex transcriptional control. Indeed, expression of hmuSTUV in the presence of inorganic iron, but not in the presence of hemin, requires the global regulator IscR acting from a promoter in the intergenic region between hmuR and hmuS. This effect of IscR appears to be direct by binding a site mapped by DNaseI footprinting. In contrast, expression of hmuR under iron limiting conditions requires derepression of the ferric uptake regulator Fur acting from the hmuR promoter, as Fur binding upstream of hmuR was demonstrated biochemically. Differential expression by both Fur and IscR would facilitate maximal hemin uptake and utilization when iron and heme availability is low while maintaining the capacity for periplasmic removal and cytosolic detoxification of heme under a wider variety of conditions. We also demonstrate that a Y. pseudotuberculosis ΔiscR mutant has a survival defect when incubated in whole blood, in which iron is sequestered by heme-containing proteins. Surprisingly, this phenotype was independent of the Hmu system, the type III secretion system, complement, and the ability of Yersinia to replicate intracellularly. These results suggest that IscR regulates multiple virulence factors important for Yersinia survival and growth in mammalian tissues and reveal a surprising complexity of heme uptake expression and function under differing conditions of iron.