IU Indianapolis ScholarWorks :: Browsing by Author "Spinola, Stanley M."

Browsing by Author "Spinola, Stanley M."

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A Haemophilus ducreyi strain lacking the yfeABCD iron transport system is virulent in human volunteers
(American Society for Microbiology, 2024) Fortney, Kate R.; Brothwell, Julie A.; Batteiger, Teresa A.; Duplantier, Rory; Katz, Barry P.; Spinola, Stanley M.; Medicine, School of Medicine
Haemophilus ducreyi causes the genital ulcer disease chancroid and painful cutaneous ulcers in children who live in the tropics. To acquire heme from the host, H. ducreyi expresses a TonB-dependent hemoglobin receptor, HgbA, which is necessary and sufficient for H. ducreyi to progress to the pustular stage of disease in a controlled human infection model. HgbA transports hemoglobin across the outer membrane; how heme is transported across the cytoplasmic membrane is unclear. In previous studies, transcripts encoding the YfeABCD heme transporter were upregulated in experimental lesions caused by H. ducreyi in human volunteers, suggesting the latter may have a role in virulence. Here we constructed a double deletion mutant, 35000HPΔyfeABΔyfeCD, which exhibited growth defects relative to its parent 35000HP in media containing human hemoglobin as an iron source. Five human volunteers were inoculated at three sites on the skin overlying the deltoid with each strain. The results of the trial showed that papules formed at 100% (95% CI, 71.5, 100) at both 35000HP and 35000HPΔyfeABΔyfeCD-inoculated sites (P = 1.0). Pustules formed at 60% (95% CI, 25.9, 94.1) at parent-inoculated sites and 53% (95% CI, 18.3, 88.4) at mutant-inoculated sites (P = 0.79). Thus, the ABC transporter encoded by yfeAB and yfeCD was dispensable for H. ducreyi virulence in humans. In the absence of YfeABCD, H. ducreyi likely utilizes other periplasmic binding proteins and ABC-transporters such as HbpA, SapABCDF, and DppBCDF to shuttle heme from the periplasm into the cytoplasm, underscoring the importance of redundancy of such systems in gram-negative pathogens.
A high-resolution view of the immune and stromal cell response to Haemophilus ducreyi infection in human volunteers
(American Society for Microbiology, 2025) Brothwell, Julie A.; Wei, Yuhui; Wang, Jia; Guo, Tingbo; Zhang, Chi; Fortney, Kate R.; Duplantier, Rory; Chen, Li; Batteiger, Teresa A.; Kaplan, Mark H.; Spinola, Stanley M.; Cao, Sha; Microbiology and Immunology, School of Medicine
Haemophilus ducreyi causes the genital ulcer disease chancroid and cutaneous ulcers in children. To study its pathogenesis, we developed a human challenge model in which we infect the skin on the upper arm of human volunteers with H. ducreyi to the pustular stage of disease. The model has been used to define lesional architecture, describe the immune infiltrate into the infected sites using flow cytometry, and explore the molecular basis of the immune response using bulk RNA-seq. Here, we used single cell RNA-seq (scRNA-seq) and spatial transcriptomics to simultaneously characterize multiple cell types within infected human skin and determine the cellular origin of differentially expressed transcripts that we had previously identified by bulk RNA-seq. We obtained paired biopsies of pustules and wounded (mock infected) sites from five volunteers for scRNA-seq. We identified 13 major cell types, including T- and NK-like cells, macrophages, dendritic cells, as well as other cell types typically found in the skin. Immune cell types were enriched in pustules, and some subtypes within the major cell types were exclusive to pustules. Sufficient tissue specimens for spatial transcriptomics were available from four of the volunteers. T- and NK-like cells were highly associated with multiple antigen presentation cell types. In pustules, type I interferon stimulation was high in areas that were high in antigen presentation-especially in macrophages near the abscess-compared to wounds. Together, our data provide a high-resolution view of the cellular immune response to the infection of the skin with a human pathogen. IMPORTANCE: A high-resolution view of the immune infiltrate due to infection with an extracellular bacterial pathogen in human skin has not yet been defined. Here, we used the human skin pathogen Haemophilus ducreyi in a human challenge model to identify on a single cell level the types of cells that are present in volunteers who fail to spontaneously clear infection and form pustules. We identified 13 major cell types. Immune cells and immune-activated stromal cells were enriched in pustules compared to wounded (mock infected) sites. Pustules formed despite the expression of multiple pro-inflammatory cytokines, such as IL-1β and type I interferon. Interferon stimulation was most evident in macrophages, which were proximal to the abscess. The pro-inflammatory response within the pustule may be tempered by regulatory T cells and cells that express indoleamine 2,3-dioxygenase, leading to failure of the immune system to clear H. ducreyi.
Both MisR (CpxR) and MisS (CpxA) Are Required for Neisseria gonorrhoeae Infection in a Murine Model of Lower Genital Tract Infection
(American Society for Microbiology, 2017-08-18) Gangaiah, Dharanesh; Raterman, Erica L.; Wu, Hong; Fortney, Kate R.; Gao, Hongyu; Liu, Yunlong; Jerse, Ann E.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
During infection, Neisseria gonorrhoeae senses and responds to stress; such responses may be modulated by MisRS (NGO0177 and NGO0176), a two-component system that is a homolog of CpxRA. In Escherichia coli, CpxRA senses and responds to envelope stress; CpxA is a sensor kinase/phosphatase for CpxR, a response regulator. When a cpxA mutant is grown in medium containing glucose, CpxR is phosphorylated by acetyl phosphate but cannot be dephosphorylated, resulting in constitutive activation. Kandler and coworkers (J. L. Kandler, C. L. Holley, J. L. Reimche, V. Dhulipala, J. T. Balthazar, A. Muszyński, R. W. Carlson, and W. M. Shafer, Antimicrob Agents Chemother 60:4690-4700, 2016, https://doi.org/10.1128/AAC.00823-16) showed that MisR (CpxR) is required for the maintenance of membrane integrity and resistance to antimicrobial peptides, suggesting a role in gonococcal survival in vivo Here, we evaluated the contributions of MisR and MisS (CpxA) to gonococcal infection in a murine model of cervicovaginal colonization and identified MisR-regulated genes using RNA sequencing (RNA-Seq). The deletion of misR or misS severely reduced the capacity of N. gonorrhoeae to colonize mice or maintain infection over a 7-day period and reduced microbial fitness after exposure to heat shock. Compared to the wild type (WT), the inactivation of misR identified 157 differentially regulated genes, most of which encoded putative envelope proteins. The inactivation of misS identified 17 differentially regulated genes compared to the WT and 139 differentially regulated genes compared to the misR mutant, 111 of which overlapped those differentially expressed in the comparison of the WT versus the misR mutant. These data indicate that an intact MisRS system is required for gonococcal infection of mice. Provided the MisR is constitutively phosphorylated in the misS mutant, the data suggest that controlled but not constitutive activation is required for gonococcal infection in mice.
A Class I Haemophilus ducreyi Strain Containing a Class II hgbA Allele Is Partially Attenuated in Humans: Implications for HgbA Vaccine Efficacy Trials
(American Society for Microbiology, 2019-07) Leduc, Isabelle; Fortney, Kate R.; Janowicz, Diane M.; Zwickl, Beth; Ellinger, Sheila; Katz, Barry P.; Lin, Huaiying; Dong, Qunfeng; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
Haemophilus ducreyi causes chancroid and is a major cause of cutaneous ulcers in children. Due to environmental reservoirs, both class I and class II H. ducreyi strains persist in cutaneous ulcer regions of endemicity following mass drug administration of azithromycin, suggesting the need for a vaccine. The hemoglobin receptor (HgbA) is a leading vaccine candidate, but its efficacy in animal models is class specific. Controlled human infection models can be used to evaluate vaccines, but only a class I strain (35000HP) has been characterized in this model. As a prelude to evaluating HgbA vaccines in the human model, we tested here whether a derivative of 35000HP containing a class II hgbA allele (FX548) is as virulent as 35000HP in humans. In eight volunteers infected at three sites with each strain, the papule formation rate was 95.8% for 35000HP versus 62.5% for FX548 (P = 0.021). Excluding doses of FX548 that were ≥2-fold higher than those of 35000HP, the pustule formation rate was 25% for 35000HP versus 11.7% for FX548 (P = 0.0053). By Western blot analysis, FX548 and 35000HP expressed equivalent amounts of HgbA in whole-cell lysates and outer membranes. The growth of FX548 and 35000HP was similar in media containing hemoglobin or hemin. By whole-genome sequencing and single-nucleotide polymorphism analysis, FX548 contained no mutations in open reading frames other than hgbA. We conclude that by an unknown mechanism, FX548 is partially attenuated in humans and is not a suitable strain for HgbA vaccine efficacy trials in the model.
Corrigendum to: Human Challenge Studies Are Unlikely to Accelerate Coronavirus Vaccine Licensure due to Ethical and Practical Issues
(Oxford University Press, 2021-05-28) Spinola, Stanley M.; Zimet, Gregory D.; Ott, Mary A.; Katz, Barry P.; Microbiology and Immunology, School of Medicine
This corrects the article "Human Challenge Studies Are Unlikely to Accelerate Coronavirus Vaccine Licensure Due to Ethical and Practical Issues" in volume 222 on page 1572; https://doi.org/10.1093/infdis/jiaa457
CpxA Phosphatase Inhibitor Activates CpxRA and Is a Potential Treatment for Uropathogenic Escherichia coli in a Murine Model of Infection
(American Society for Microbiology, 2022-04-27) Fortney, Kate R.; Smith, Sara N.; van Rensburg, Julia J.; Brothwell, Julie A.; Gardner, Jessi J.; Katz, Barry P.; Ahsan, Nagib; Duerfeldt, Adam S.; Mobley, Harry L.T.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
CpxRA is an envelope stress response system that is highly conserved in the Enterobacteriaceae. CpxA has kinase activity for CpxR and phosphatase activity for phospho-CpxR (CpxR-P), a transcription factor. In response to membrane stress, CpxR-P is produced and upregulates genes involved in membrane repair and downregulates genes that encode virulence factors that are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and in uropathogenic Escherichia coli (UPEC) are attenuated in murine models. We hypothesized that pharmacologic activation of CpxR could serve as an antimicrobial/antivirulence strategy and recently showed that 2,3,4,9-tetrahydro-1H-carbazol-1-amines activate the CpxRA system by inhibiting CpxA phosphatase activity. Here, we tested the ability of a series of three CpxRA-activating compounds with increasing potency to clear UPEC stain CFT073 in a murine urinary tract infection model. We show that these compounds are well tolerated and achieve sufficient levels to activate CpxR in the kidneys, bladder, and urine. Although the first two compounds were ineffective in promoting clearance of CFT073 in the murine model, the most potent derivative, compound 26, significantly reduced bacterial recovery in the urine and trended toward reducing bacterial recovery in the bladder and kidneys, with efficacy similar to ciprofloxacin. Treatment of CFT073 cultured in human urine with compound 26 fostered accumulation of CpxR-P and decreased the expression of proteins involved in siderophore biosynthesis and binding, heme degradation, and flagellar movement. These studies suggest that chemical activation of CpxRA may present a viable strategy for treating infections due to UPEC. IMPORTANCE: The increasing prevalence of urinary tract infections (UTIs) due to antibiotic-resistant uropathogenic Escherichia coli (UPEC) is a major public health concern. Bacteria contain proteins that sense their environment and have no human homologs and, thus, are attractive drug targets. CpxRA is a conserved sensing system whose function is to reduce stress in the bacterial cell membrane; activation of CpxRA reduces the expression of virulence determinants, which must cross the cell membrane to reach the bacterial surface. We previously identified a class of compounds that activate CpxRA. We show in a mouse UTI model that our most potent compound significantly reduced recovery of UPEC in the urine, trended toward reducing bacterial recovery in the bladder and kidneys, did not kill UPEC, and downregulated multiple proteins involved in UPEC virulence. Since these compounds do not act by a killing mechanism, they have potential to treat UTIs caused by antibiotic-resistant bacteria.
Determination of an Interaction Network between an Extracellular Bacterial Pathogen and the Human Host
(American Society for Microbiology, 2019-06-18) Griesenauer, Brad; Tran, Tuan M.; Fortney, Kate R.; Janowicz, Diane M.; Johnson, Paula; Gao, Hongyu; Barnes, Stephen; Wilson, Landon S.; Liu, Yunlong; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
A major gap in understanding infectious diseases is the lack of information about molecular interaction networks between pathogens and the human host. Haemophilus ducreyi causes the genital ulcer disease chancroid in adults and is a leading cause of cutaneous ulcers in children in the tropics. We developed a model in which human volunteers are infected on the upper arm with H. ducreyi until they develop pustules. To define the H. ducreyi and human interactome, we determined bacterial and host transcriptomic and host metabolomic changes in pustules. We found that in vivo H. ducreyi transcripts were distinct from those in the inocula, as were host transcripts in pustule and wounded control sites. Many of the upregulated H. ducreyi genes were found to be involved in ascorbic acid and anaerobic metabolism and inorganic ion/nutrient transport. The top 20 significantly expressed human pathways showed that all were involved in immune responses. We generated a bipartite network for interactions between host and bacterial gene transcription; multiple positively correlated networks contained H. ducreyi genes involved in anaerobic metabolism and host genes involved with the immune response. Metabolomic studies showed that pustule and wounded samples had different metabolite compositions; the top ion pathway involved ascorbate and aldarate metabolism, which correlated with the H. ducreyi transcriptional response and upregulation of host genes involved in ascorbic acid recycling. These data show that an interactome exists between H. ducreyi and the human host and suggest that H. ducreyi exploits the metabolic niche created by the host immune response.IMPORTANCE Dual RNA sequencing (RNA-seq) offers the promise of determining an interactome at a transcriptional level between a bacterium and the host but has yet to be done on any bacterial infection in human tissue. We performed dual RNA-seq and metabolomics analyses on wounded and infected sites following experimental infection of the arm with H. ducreyi Our results suggest that H. ducreyi survives in an abscess by utilizing l-ascorbate as an alternative carbon source, possibly taking advantage of host ascorbic acid recycling, and that H. ducreyi also adapts by upregulating genes involved in anaerobic metabolism and inorganic ion and nutrient transport. To our knowledge, this is the first description of an interaction network between a bacterium and the human host at a site of infection.
Development and validation of a high-throughput cell-based screen to identify activators of a bacterial two-component signal transduction system
(ACC, 2015-07) van Rensburg, Julia J.; Fortney, Kate R.; Chen, Lan; Krieger, Andrew J.; Lima, Bruno P.; Wolfe, Alan J.; Katz, Barry P.; Zhang, Zhong-Yin; Spinola, Stanley M.; Department of Microbiology and Immunology, IU School of Medicine
CpxRA is a two-component signal transduction system (2CSTS) found in many drug-resistant Gram-negative bacteria. In response to periplasmic stress, CpxA autophosphorylates and donates a phosphoryl group to its cognate response regulator, CpxR. Phosphorylated CpxR (CpxR-P) upregulates genes involved in membrane repair and downregulates multiple genes that encode virulence factors, which are trafficked across the cell membrane. Mutants that constitutively activate CpxRA in Salmonella enterica serovar Typhimurium and Haemophilus ducreyi are avirulent in mice and humans, respectively. Thus, the activation of CpxRA has high potential as a novel antimicrobial/antivirulence strategy. Using a series of Escherichia coli strains containing a CpxR-P-responsive lacZ reporter and deletions in genes encoding CpxRA system components, we developed and validated a novel cell-based high-throughput screen (HTS) for CpxRA activators. A screen of 36,000 compounds yielded one hit compound that increased reporter activity in wild-type cells. This is the first report of a compound that activates, rather than inhibits, a 2CSTS. The activity profile of the compound against CpxRA pathway mutants in the presence of glucose suggested that the compound inhibits CpxA phosphatase activity. We confirmed that the compound induced the accumulation of CpxR-P in treated cells. Although the hit compound contained a nitro group, a derivative lacking this group retained activity in serum and had lower cytotoxicity than that of the initial hit. This HTS is amenable for the screening of larger libraries to find compounds that activate CpxRA by other mechanisms, and it could be adapted to find activators of other two-component systems.
Dispensability of Ascorbic Acid Uptake and Utilization Encoded by ulaABCD for the Virulence of Haemophilus ducreyi in Humans
(Oxford University Press, 2023) Brothwell, Julie A.; Fortney, Kate R.; Batteiger, Teresa; Katz, Barry P.; Spinola, Stanley M.; Microbiology and Immunology, School of Medicine
Compared with wounded skin, ascorbic acid is enriched in pustules of humans experimentally infected with Haemophilus ducreyi. Compared with the broth-grown inocula, transcription of the H. ducreyi ulaABCD operon, which encodes genes for ascorbic acid uptake, is increased in pustules. We hypothesized that ascorbic acid uptake plays a role in H. ducreyi virulence. Five volunteers were infected with both H. ducreyi strain 35000HP and its isogenic ulaABCD deletion mutant at multiple sites; the papule and pustule formation rates of the mutant and parent strains were similar. Thus, ascorbic acid uptake is not essential for H. ducreyi virulence in humans.
Draft Whole-Genome Sequence of Haemophilus ducreyi Strain AUSPNG1, Isolated from a Cutaneous Ulcer of a Child from Papua New Guinea.
(ASM, 2016-02) Gangaiah, Dharanesh; Marinov, Georgi K.; Roberts, Sally A.; Robson, Jenny; Spinola, Stanley M.; Department of Microbiology & Immunology, IU School of Medicine
Haemophilus ducreyi has recently emerged as a leading cause of cutaneous ulcers in the yaws-endemic areas of Papua New Guinea and other South Pacific islands. Here, we report the draft genome sequence of the H. ducreyi strain AUSPNG1, isolated from a cutaneous ulcer of a child from Papua New Guinea.

Browsing by Author "Spinola, Stanley M."

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