Two Methods of Measuring Cryptococcus neoformans Fungal Burden in Macrophages.

The ability of C. neoformans to survive and replicate within host phagocytes enables it to evade the immune system and allows for persistence of the infection. As such, measuring fungal burden of C. neoformans strains-and indeed how drug treatments can influence fungal burden-provides important information about C. neoformans pathogenesis. In this chapter, we describe two methods that may be used to appraise fungal burden: a standard end-point colony-formation assay for calculating the average number of yeast per host cell and a fluorescence microscopy-based method that may be used to measure changes in fungal burden in individual living macrophages in real time.

Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

Exploring the role of CITED transcriptional regulators in the control of macrophage polarization.

Macrophages are tissue resident innate phagocytic cells that take on contrasting phenotypes, or polarization states, in response to the changing combination of microbial and cytokine signals at sites of infection. During the opening stages of an infection, macrophages adopt the proinflammatory, highly antimicrobial M1 state, later shifting to an anti-inflammatory, pro-tissue repair M2 state as the infection resolves. The changes in gene expression underlying these transitions are primarily governed by nuclear factor kappaB (NF-κB), Janus kinase (JAK)/signal transducer and activation of transcription (STAT), and hypoxia-inducible factor 1 (HIF1) transcription factors, the activity of which must be carefully controlled to ensure an effective yet spatially and temporally restricted inflammatory response. While much of this control is provided by pathway-specific feedback loops, recent work has shown that the transcriptional co-regulators of the CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxy-terminal domain (CITED) family serve as common controllers for these pathways. In this review, we describe how CITED proteins regulate polarization-associated gene expression changes by controlling the ability of transcription factors to form chromatin complexes with the histone acetyltransferase, CBP/p300. We will also cover how differences in the interactions between CITED1 and 2 with CBP/p300 drive their contrasting effects on pro-inflammatory gene expression.

Clinical Presentation of Small Intestinal Bacterial Overgrowth from Aerodigestive Tract Bacteria Versus Colonic-Type Bacteria: A Comparison Study.

BACKGROUND: Small Intestinal Bacterial Overgrowth (SIBO) is a heterogenous syndrome from excessive bacteria in the small intestine lumen. It is unknown if differences in type of bacterial overgrowth lead to differences in symptoms. METHODS: Patients with suspected SIBO were recruited prospectively. Exclusion criteria were probiotics, antibiotics, or bowel prep in preceding 30 days. Clinical characteristics, risk factors, and labs were collected. Proximal jejunal aspiration via upper enteroscopy was performed. Aerodigestive tract (ADT) SIBO was defined as > 105 CFU/mL of oropharyngeal and respiratory bacteria. Colonic-type SIBO was defined as > 104 CFU/mL of distal small bowel and colon bacteria. Aims were to compare symptom profiles, clinical complications, labs, and underlying risk factors between ADT and colonic-type SIBO. KEY RESULTS: We consented 166 subjects. Aspiration was not obtained in 22 and SIBO was found in 69 (49%) of 144 subjects. Daily abdominal distention trended towards more prevalent in ADT SIBO versus colonic-type SIBO (65.2% vs 39.1%, p = 0.09). Patient symptom scores were similar. Iron deficiency was more prevalent in ADT SIBO (33.3% vs 10.3%, p = 0.04). Subjects with colonic-type SIBO were more likely to have a risk factor for colonic bacteria colonization (60.9% vs 17.4%, p = 0.0006). Subjects with ADT SIBO were more likely to have a risk factor for diminished gastric acid (91.3% vs 67.4%, p = 0.02). CONCLUSIONS & INFERENCES: We found differences in iron deficiency and underlying risk factors between ADT and colonic-type SIBO. However, distinct clinical profiles remained elusive. Future research is needed to develop validated symptom assessment tools and distinguish cause from correlation.

Characterization of Vaginal Microbial Community Dynamics in the Pathogenesis of Incident Bacterial Vaginosis, a Pilot Study.

BACKGROUND: Despite more than 60 years of research, the etiology of bacterial vaginosis (BV) remains controversial. In this pilot study, we used shotgun metagenomic sequencing to characterize vaginal microbial community changes before the development of incident BV (iBV). METHODS: A cohort of African American women with a baseline healthy vaginal microbiome (no Amsel criteria, Nugent score 0-3 with no Gardnerella vaginalis morphotypes) were followed for 90 days with daily self-collected vaginal specimens for iBV (≥2 consecutive days of a Nugent score of 7-10). Shotgun metagenomic sequencing was performed on select vaginal specimens from 4 women, every other day for 12 days before iBV diagnosis. Sequencing data were analyzed through Kraken2 and bioBakery 3 workflows, and specimens were classified into community state types. Quantitative polymerase chain reaction was performed to compare the correlation of read counts with bacterial abundance. RESULTS: Common BV-associated bacteria such as G. vaginalis , Prevotella bivia , and Fannyhessea vaginae were increasingly identified in the participants before iBV. Linear modeling indicated significant increases in G. vaginalis and F . vaginae relative abundance before iBV, whereas the relative abundance of Lactobacillus species declined over time. The Lactobacillus species decline correlated with the presence of Lactobacillus phages. We observed enrichment in bacterial adhesion factor genes on days before iBV. There were also significant correlations between bacterial read counts and abundances measured by quantitative polymerase chain reaction. CONCLUSIONS: This pilot study characterizes vaginal community dynamics before iBV and identifies key bacterial taxa and mechanisms potentially involved in the pathogenesis of iBV.

Sexual behavior shapes male genitourinary microbiome composition.

The origin, composition, and significance of the distal male urethral microbiome are unclear, but vaginal microbiome dysbiosis is linked to new sex partners and several urogynecological syndromes. We characterized 110 urethral specimens from men without urethral symptoms, infections, or inflammation using shotgun metagenomics. Most urethral specimens contain characteristic lactic acid bacteria and Corynebacterium spp. In contrast, several bacteria associated with vaginal dysbiosis were present only in specimens from men who reported vaginal intercourse. Sexual behavior, but not other evaluated behavioral, demographic, or clinical variables, strongly associated with inter-specimen variance in urethral microbiome composition. Thus, the male urethra supports a simple core microbiome that is established independent of sexual exposures but can be re-shaped by vaginal sex. Overall, the results suggest that urogenital microbiology and sexual behavior are inexorably intertwined, and show that the male urethra harbors female urogenital pathobionts.

A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion.

Many obligate intracellular bacteria, including members of the genus Chlamydia, cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225, a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens (Coxiella burnetii and Buchnera aphidicola) are sufficient to import valine into Escherichia coli. We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.

Regulation of macrophage IFNγ-stimulated gene expression by the transcriptional coregulator CITED1.

Macrophages serve as a first line of defense against microbial pathogens. Exposure to interferon-γ (IFNγ) increases interferon-stimulated gene (ISG) expression in these cells, resulting in enhanced antimicrobial and proinflammatory activity. Although this response must be sufficiently vigorous to ensure the successful clearance of pathogens, it must also be carefully regulated to prevent tissue damage. This is controlled in part by CBP/p300-interacting transactivator with glutamic acid/aspartic acid-rich carboxyl-terminal domain 2 (CITED2), a transcriptional coregulator that limits ISG expression by inhibiting STAT1 and IRF1. Here, we show that the closely related Cited1 is an ISG, which is expressed in a STAT1-dependent manner, and that IFNγ stimulates the nuclear accumulation of CITED1 protein. In contrast to CITED2, ectopic CITED1 enhanced the expression of a subset of ISGs, including Ccl2, Ifit3b, Isg15 and Oas2. This effect was reversed in a Cited1-null cell line produced by CRISPR-based genomic editing. Collectively, these data show that CITED1 maintains proinflammatory gene expression during periods of prolonged IFNγ exposure and suggest that there is an antagonistic relationship between CITED proteins in the regulation of macrophage inflammatory function. This article has an associated First Person interview with the first author of the paper.

Characterization of Virulence-Associated Traits in Mycoplasma penetrans Strains Acting as Likely Etiological Agents of Idiopathic Nongonococcal Urethritis.

Mycoplasma penetrans is an emerging pathogen with a reduced genome. This bacterium has only previously been cultured from individuals with chronic immunodeficiencies. Here we report the characteristics of 4 M. penetrans isolates from the urine of immunocompetent males with nongonococcal urethritis, in comparison with strain HF-2 from an immunocompromised patient. Several features exhibited distinct differences between these isolates and HF-2. Unlike HF-2, all 4 were resistant to azithromycin. They exhibited greater sialic acid-dependent binding to erythrocytes, gliding motility speed, and H2O2 production than HF-2. All new isolates produced thinner capsules than HF-2. Invasiveness varied, with some isolates being more invasive than HF-2 and some less invasive. Cytotoxicity to HeLa cells was similar to HF-2, and all strains could clear extracellular traps produced by innate immune cells. We conclude that subtle differences among M. penetrans strains may be critical for this organism to establish an infection in an otherwise healthy individual.

Two Streptococcus pyogenes emm types and several anaerobic bacterial species are associated with idiopathic cutaneous ulcers in children after community-based mass treatment with azithromycin.

BACKGROUND: In yaws-endemic areas, two-thirds of exudative cutaneous ulcers (CU) are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD); one-third are classified as idiopathic ulcers (IU). A yaws eradication campaign on Lihir Island in Papua New Guinea utilizing mass drug administration (MDA) of azithromycin initially reduced but failed to eradicate yaws; IU rates remained constant throughout the study. Using 16S rRNA gene sequencing, we previously determined that Streptococcus pyogenes was associated with some cases of IU. Here, we applied shotgun metagenomics to the same samples we analyzed previously by 16S rRNA sequencing to verify this result, identify additional IU-associated microorganisms, and determine why S. pyogenes-associated IU might have persisted after MDA of azithromycin. METHODOLOGY/PRINCIPAL FINDINGS: We sequenced DNA extracted from 244 CU specimens separated into four groups based upon microorganism-specific PCR results (HD+, TP+, TP+HD+, and TP-HD- or IU). S. pyogenes was enriched in IU (24.71% relative abundance [RA]) specimens compared to other ulcer sub-groups, confirming our prior results. We bioinformatically identified the emm (M protein gene) types found in the S. pyogenes IU specimens and found matches to emm156 and emm166. Only ~39% of IU specimens contained detectable S. pyogenes, suggesting that additional organisms could be associated with IU. In the sub-set of S. pyogenes-negative IU specimens, Criibacterium bergeronii, a member of the Peptostreptococcaceae, and Fusobacterium necrophorum (7.07% versus 0.00% RA and 2.18% versus 0.00% RA, respectively), were enriched compared to the S. pyogenes-positive sub-set. Although a broad range of viruses were detected in the CU specimens, none were specifically associated with IU. CONCLUSIONS/SIGNIFICANCE: Our observations confirm the association of S. pyogenes with IU in yaws-endemic areas, and suggest that additional anaerobic bacteria, but not other microorganisms, may be associated with this syndrome. Our results should aid in the design of diagnostic tests and selective therapies for CU.

Esophageal Microbiome in Healthy Children and Esophageal Eosinophilia.

OBJECTIVES: There is limited knowledge about the role of esophageal microbiome in pediatric esophageal eosinophilia (EE). We aimed to characterize the esophageal microbiome in pediatric patients with and without EE. METHODS: In the present prospective study, esophageal mucosal biopsies were obtained from 41 children. Of these, 22 had normal esophageal mucosal biopsies ("healthy"), 6 children had reflux esophagitis (RE), 4 had proton pump inhibitor (PPi)-responsive esophageal eosinophilia (PPi-REE), and 9 had eosinophilic esophagitis (EoE). The microbiome composition was analyzed using 16S rRNA gene sequencing. The age median (range) in years for the healthy, RE, PPi-REE, and EoE group were 10 (1.5-18), 6 (2-15), 6.5 (5-15), and 9 (1.5-17), respectively. RESULTS: The bacterial phylum Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria were the most predominant. The Epsilonproteobacteria, Betaproteobacteria, Flavobacteria, Fusobacteria, and Sphingobacteria class were underrepresented across groups. The Vibrionales was predominant in healthy and EoE group but lower in RE and PPi-REE groups. The genus Streptococcus, Rahnella, and Leptotrichia explained 29.65% of the variation in the data with an additional 10.86% variation in the data was explained by Microbacterium, Prevotella, and Vibrio genus. The healthy group had a higher diversity and richness index compared to other groups, but this was not statistically different. CONCLUSIONS: The pediatric esophagus has an abundant and diverse microbiome, both in the healthy and diseased states. The healthy group had a higher, but not significantly different, diversity and richness index compared to other groups.

Evaluation of Clinical, Gram Stain, and Microbiological Cure Outcomes in Men Receiving Azithromycin for Acute Nongonococcal Urethritis: Discordant Cures Are Associated With Mycoplasma genitalium Infection.

BACKGROUND: In men with nongonococcal urethritis (NGU), clinicians and patients rely on clinical cure to guide the need for additional testing/treatment and when to resume sex, respectively; however, discordant clinical and microbiological cure outcomes do occur. How accurately clinical cure reflects microbiological cure in specific sexually transmitted infections (STIs) is unclear. METHODS: Men with NGU were tested for Neisseria gonorrhoeae, Chlamydia trachomatis (CT), Mycoplasma genitalium (MG), Trichomonas vaginalis, urethrotropic Neisseria meningitidis ST-11 clade strains, and Ureaplasma urealyticum (UU). Men received azithromycin 1 g and returned for a 1-month test-of-cure visit. In MG infections, we evaluated for the presence of macrolide resistance-mediating mutations (MRMs) and investigated alternate hypotheses for microbiological treatment failure using in situ shotgun metagenomic sequencing, phylogenetic analysis, multilocus sequence typing analyses, and quantitative PCR. RESULTS: Of 280 men with NGU, 121 were included in this analysis. In the monoinfection group, 52 had CT, 16 had MG, 7 had UU, 10 had mixed infection, and 36 men had idiopathic NGU. Clinical cure rates were 85% for CT, 100% for UU, 50% for MG, and 67% for idiopathic NGU. Clinical cure accurately predicted microbiological cure for all STIs, except MG. Discordant results were significantly associated with MG-NGU and predominantly reflected microbiological failure in men with clinical cure. Mycoplasma genitalium MRMs, but not MG load or strain, were strongly associated with microbiological failure. CONCLUSIONS: In azithromycin-treated NGU, clinical cure predicts microbiological cure for all STIs, except MG. Nongonococcal urethritis management should include MG testing and confirmation of microbiological cure in azithromycin-treated MG-NGU when MRM testing is unavailable.

The growing repertoire of genetic tools for dissecting chlamydial pathogenesis.

Multiple species of obligate intracellular bacteria in the genus Chlamydia are important veterinary and/or human pathogens. These pathogens all share similar biphasic developmental cycles and transition between intracellular vegetative reticulate bodies and infectious elementary forms, but vary substantially in their host preferences and pathogenic potential. A lack of tools for genetic engineering of these organisms has long been an impediment to the study of their biology and pathogenesis. However, the refinement of approaches developed in C. trachomatis over the last 10 years, and adaptation of some of these approaches to other Chlamydia spp. in just the last few years, has opened exciting new possibilities for studying this ubiquitous group of important pathogens.

Associations Between Dysmenorrhea Symptom-Based Phenotypes and Vaginal Microbiome: A Pilot Study.

BACKGROUND: Dysmenorrhea is highly prevalent; it places women at risk for other chronic pain conditions. There is a high degree of individual variability in menstrual pain severity, the number of painful sites, and co-occurring gastrointestinal symptoms. Distinct dysmenorrhea symptom-based phenotypes were previously identified, but the biological underpinnings of these phenotypes are less known. One underexplored contributor is the vaginal microbiome. The vaginal microbiota differs significantly among reproductive-age women and may modulate as well as amplify reproductive tract inflammation, which may contribute to dysmenorrhea symptoms. OBJECTIVES: The objective of this study was to examine associations between dysmenorrhea symptom-based phenotypes and vaginal microbiome compositions on- and off-menses. METHODS: We conducted a prospective, longitudinal, pilot study of 20 women (aged 15-24 years) grouped into three dysmenorrhea symptom-based phenotypes: "mild localized pain," "severe localized pain," and "severe multiple pain and gastrointestinal symptoms." Over one menstrual cycle, participants provided vaginal swabs when they were on- and off-menses. We assayed the vaginal microbiome using 16S rRNA gene sequencing. Permutational multivariate analysis of variance tests were used to compare microbiome compositions across phenotypes, with heat maps generated to visualize the relative abundance of bacterial taxa. RESULTS: The vaginal microbiome compositions (n = 40) were different across the three phenotypes. After separating the on-menses (n = 20) and off-menses (n = 20) specimens, the statistically significant difference was seen on-menses, but not off-menses. Compared to the "mild localized pain" phenotype, participants in the "multiple severe symptoms" phenotype had a lower lactobacilli level and a higher abundance of Prevotella, Atopobium, and Gardnerella when on-menses. We also observed trends of differences across phenotypes in vaginal microbiome change from off- to on-menses. DISCUSSION: The study provides proof-of-concept data to support larger studies on associations between dysmenorrhea symptom-based phenotypes and vaginal microbiome that might lead to new intervention targets and/or biomarkers for dysmenorrhea. This line of research has the potential to inform precision dysmenorrhea treatment that can improve women's quality of life.

A Same-Genus Screening Approach Reveals Novel Effectors and New Possibilities for Investigating Chlamydia Pathogenesis.

Chlamydiae are obligate intracellular pathogens that rely on secreted effector proteins to establish their intracellular niche. In this issue of the Journal of Bacteriology, Yanatori et al describe a screen for C. pneumoniae effectors, performed in C. trachomatis, which identified several new proteins that are translocated during infection (Yanatori, Miura et al. 2021). More broadly, they demonstrate how new genetic approaches in C. trachomatis can be used to characterize the virulence factors of other Chlamydia species.

Genome copy number regulates inclusion expansion, septation, and infectious developmental form conversion in Chlamydia trachomatis.

DNA replication is essential for the growth and development of Chlamydia trachomatis, however it is unclear how this process contributes to and is controlled by the pathogen's biphasic lifecycle. While inhibitors of transcription, translation, cell division, and glucose-6-phosphate transport all negatively affect chlamydial intracellular development, the effects of directly inhibiting DNA polymerase have never been examined. We isolated a temperature sensitive dnaE mutant (dnaEts ) that exhibits a ∼100-fold reduction in genome copy number at the non-permissive temperature (40°C), but replicates similarly to the parent at the permissive temperature of 37°C. We measured higher ratios of genomic DNA nearer the origin of replication than the terminus in dnaEts at 40°C, indicating that this replication deficiency is due to a defect in DNA polymerase processivity. dnaEts formed fewer and smaller pathogenic vacuoles (inclusions) at 40°C, and the bacteria appeared enlarged and exhibited defects in cell division. The bacteria also lacked both discernable peptidoglycan and polymerized MreB, the major cell division organizing protein in Chlamydia responsible for nascent peptidoglycan biosynthesis. We also found that absolute genome copy number, rather than active genome replication, was sufficient for infectious progeny production. Deficiencies in both genome replication and inclusion expansion reversed when dnaEts was shifted from 40°C to 37°C early in infection, and intragenic suppressor mutations in dnaE also restored dnaEts genome replication and inclusion expansion at 40°C. Overall, our results show that genome replication in C. trachomatis is required for inclusion expansion, septum formation, and the transition between the microbe's replicative and infectious forms.SIGNIFICANCE Chlamydiae transition between infectious, extracellular elementary bodies (EBs) and non-infectious, intracellular reticulate bodies (RBs). Some checkpoints that govern transitions in chlamydial development have been identified, but the extent to which genome replication plays a role in regulating the pathogen's infectious cycle has not been characterized. We show that genome replication is dispensable for EB to RB conversion, but is necessary for RB proliferation, division septum formation, and inclusion expansion. We use new methods to investigate developmental checkpoints and dependencies in Chlamydia that facilitate the ordering of events in the microbe's biphasic life cycle. Our findings suggest that Chlamydia utilizes feedback inhibition to regulate core metabolic processes during development, likely an adaptation to intracellular stress and a nutrient-limiting environment.

Streptococcus pyogenes Is Associated with Idiopathic Cutaneous Ulcers in Children on a Yaws-Endemic Island.

Exudative cutaneous ulcers (CU) in yaws-endemic areas are associated with Treponema pallidum subsp. pertenue (TP) and Haemophilus ducreyi (HD), but one-third of CU cases are idiopathic (IU). Using mass drug administration (MDA) of azithromycin, a yaws eradication campaign on Lihir Island in Papua New Guinea reduced but failed to eradicate yaws; IU rates remained constant throughout the campaign. To identify potential etiologies of IU, we obtained swabs of CU lesions (n = 279) and of the skin of asymptomatic controls (AC; n = 233) from the Lihir Island cohort and characterized their microbiomes using a metagenomics approach. CU bacterial communities were less diverse than those of the AC. Using real-time multiplex PCR with pathogen-specific primers, we separated CU specimens into HD-positive (HD+), TP+, HD+TP+, and IU groups. Each CU subgroup formed a distinct bacterial community, defined by the species detected and/or the relative abundances of species within each group. Streptococcus pyogenes was the most abundant organism in IU (22.65%) and was enriched in IU compared to other ulcer groups. Follow-up samples (n = 31) were obtained from nonhealed ulcers; the average relative abundance of S. pyogenes was 30.11% in not improved ulcers and 0.88% in improved ulcers, suggesting that S. pyogenes in the not improved ulcers may be azithromycin resistant. Catonella morbi was enriched in IU that lacked S. pyogenes As some S. pyogenes and TP strains are macrolide resistant, penicillin may be the drug of choice for CU azithromycin treatment failures. Our study will aid in the design of diagnostic tests and selective therapies for CU.IMPORTANCE Cutaneous ulcers (CU) affect approximately 100,000 children in the tropics each year. While two-thirds of CU are caused by Treponema pallidum subspecies pertenue and Haemophilus ducreyi, the cause(s) of the remaining one-third is unknown. Given the failure of mass drug administration of azithromycin to eradicate CU, the World Health Organization recently proposed an integrated disease management strategy to control CU. Success of this strategy requires determining the unknown cause(s) of CU. By using 16S rRNA gene sequencing of swabs obtained from CU and the skin of asymptomatic children, we identified another possible cause of skin ulcers, Streptococcus pyogenes Although S. pyogenes is known to cause impetigo and cellulitis, this is the first report implicating the organism as a causal agent of CU. Inclusion of S. pyogenes into the integrated disease management plan will improve diagnostic testing and treatment of this painful and debilitating disease of children and strengthen elimination efforts.

A Genital Infection-Attenuated Chlamydia muridarum Mutant Infects the Gastrointestinal Tract and Protects against Genital Tract Challenge.

Chlamydia spp. productively infect mucosal epithelial cells of multiple anatomical sites, including the conjunctiva, lungs, gastrointestinal (GI) tract, and urogenital tract. We, and others, previously established that chlamydial GI tropism is mediated by distinct chromosomal and plasmid factors. In this study, we describe a genital infection-attenuated Chlamydia muridarum mutant (GIAM-1) that is profoundly and specifically attenuated in the murine genital tract. GIAM-1 infected the murine GI tract similarly to wild-type (WT) Chlamydia muridarum but did not productively infect the lower genital tract of female mice, ascend to infect the upper genital tract, or cause hydrosalpinx. However, GI infection of mice with GIAM-1 elicited a transmucosal immune response that protected against subsequent genital challenge with WT Chlamydia muridarum Collectively, our results demonstrate that chlamydia mutants that are profoundly attenuated for specific organ tissues can be derived and demonstrate that live-attenuated vaccine strains that infect the GI tract, but do not elicit genital tract disease, could be used to protect against chlamydia genital tract infection and disease.IMPORTANCE Chlamydia is the most common sexually transmitted bacterial infection in the United States. Most chlamydia genital infections resolve without serious consequences; however, untreated infection in women can cause pelvic inflammatory disease and infertility. Antibiotics are very effective in treating chlamydia, but most genital infections in both men and women are asymptomatic and go undiagnosed. Therefore, there is a critical need for an effective vaccine. In this work, we show that a mutant chlamydia strain, having substantially reduced virulence for genital infection, colonizes the gastrointestinal tract and produces robust immunity to genital challenge with fully virulent wild-type chlamydia. These results are an important advance in understanding chlamydial virulence and provide compelling evidence that safe and effective live-attenuated chlamydia vaccines may be feasible.

Intracellular Cryptococcus neoformans disrupts the transcriptome profile of M1- and M2-polarized host macrophages.

Macrophages serve as a first line of defense against infection with the facultative intracellular pathogen, Cryptococcus neoformans (Cn). However, the ability of these innate phagocytic cells to destroy ingested Cn is strongly influenced by polarization state with classically (M1) activated macrophages better able to control cryptococcal infections than alternatively (M2) activated cells. While earlier studies have demonstrated that intracellular Cn minimally affects the expression of M1 and M2 markers, the impact on the broader transcriptome associated with these states remains unclear. To investigate this, an in vitro cell culture model of intracellular infection together with RNA sequencing-based transcriptome profiling was used to measure the impact of Cn infection on gene expression in both polarization states. The gene expression profile of both M1 and M2 cells was extensively altered to become more like naive (M0) macrophages. Gene ontology analysis suggested that this involved changes in the activity of the Janus kinase-signal transducers and activators of transcription (JAK-STAT), p53, and nuclear factor-κB (NF-κB) pathways. Analyses of the principle polarization markers at the protein-level also revealed discrepancies between the RNA- and protein-level responses. In contrast to earlier studies, intracellular Cn was found to increase protein levels of the M1 marker iNos. In addition, common gene expression changes were identified that occurred post-Cn infection, independent of polarization state. This included upregulation of the transcriptional co-regulator Cited1, which was also apparent at the protein level in M1-polarized macrophages. These changes constitute a transcriptional signature of macrophage Cn infection and provide new insights into how Cn impacts gene expression and the phenotype of host phagocytes.