Phase-variable bacteria simultaneously express multiple capsules.

Capsular polysaccharides (CPSs) protect bacteria from host and environmental factors. Many bacteria can express different CPSs and these CPSs are phase variable. For example, Bacteroides thetaiotaomicron (B. theta) is a prominent member of the human gut microbiome and expresses eight different capsular polysaccharides. Bacteria, including B. theta, have been shown to change their CPSs to adapt to various niches such as immune, bacteriophage, and antibiotic perturbations. However, there are limited tools to study CPSs and fundamental questions regarding phase variance, including if gut bacteria can express more than one capsule at the same time, remain unanswered. To better understand the roles of different CPSs, we generated a B. theta CPS1-specific antibody and a flow cytometry assay to detect CPS expression in individual bacteria in the gut microbiota. Using these novel tools, we report for the first time that bacteria can simultaneously express multiple CPSs. We also observed that nutrients such as glucose and salts had no effect on CPS expression. The ability to express multiple CPSs at the same time may provide bacteria with an adaptive advantage to thrive amid changing host and environmental conditions, especially in the intestine.

Immunomodulatory Roles of Polysaccharide Capsules in the Intestine.

The interplay between the immune system and the microbiota in the human intestine dictates states of health vs. disease. Polysaccharide capsules are critical elements of bacteria that protect bacteria against environmental and host factors, including the host immune system. This review summarizes the mechanisms by which polysaccharide capsules from commensal and pathogenic bacteria in the gut microbiota modulate the innate and adaptive immune systems in the intestine. A deeper understanding of the roles of polysaccharide capsules in microbiota-immune interactions will provide a basis to harness their therapeutic potential to advance human health.

Polysaccharide Capsules Equip the Human Symbiont Bacteroides thetaiotaomicron to Modulate Immune Responses to a Dominant Antigen in the Intestine.

Bacteria express multiple diverse capsular polysaccharides (CPSs) for protection against environmental and host factors, including the host immune system. Using a mouse TCR transgenic CD4+ T cell, BθOM, that is specific for B. thetaiotaomicron and a complete set of single CPS-expressing B. thetaiotaomicron strains, we ask whether CPSs can modify the immune responses to specific bacterial Ags. Acapsular B. thetaiotaomicron, which lacks all B. thetaiotaomicron CPSs, stimulated BθOM T cells more strongly than wild-type B. thetaiotaomicron Despite similar levels of BθOM Ag expression, many single CPS-expressing B. thetaiotaomicron strains were antistimulatory and weakly activated BθOM T cells, but a few strains were prostimulatory and strongly activated BθOM T cells just as well or better than an acapsular strain. B. thetaiotaomicron strains that expressed an antistimulatory CPS blocked Ag delivery to the immune system, which could be rescued by Fc receptor-dependent Ab opsonization. All single CPS-expressing B. thetaiotaomicron strains stimulated the innate immune system to skew toward M1 macrophages and release inflammatory cytokines in an MyD88-dependent manner, with antistimulatory CPS activating the innate immune system in a weaker manner than prostimulatory CPS. The expression of antistimulatory versus prostimulatory CPSs on outer membrane vesicles also regulated immune responses. Moreover, antistimulatory and prostimulatory single CPS-expressing B. thetaiotaomicron strains regulated the activation of Ag-specific and polyclonal T cells as well as clearance of dominant Ag in vivo. These studies establish that the immune responses to specific bacterial Ags can be modulated by a diverse set of CPSs.

Diet modulates colonic T cell responses by regulating the expression of a Bacteroides thetaiotaomicron antigen.

T cell responses to symbionts in the intestine drive tolerance or inflammation depending on the genetic background of the host. These symbionts in the gut sense the available nutrients and adapt their metabolic programs to use these nutrients efficiently. Here, we ask whether diet can alter the expression of a bacterial antigen to modulate adaptive immune responses. We generated a CD4+ T cell hybridoma, BθOM, specific for Bacteroides thetaiotaomicron (B. theta). Adoptively transferred transgenic T cells expressing the BθOM TCR proliferated in the colon, colon-draining lymph node, and spleen in B. theta-colonized healthy mice and differentiated into regulatory T cells (Tregs) and effector T cells (Teffs). Depletion of B. theta-specific Tregs resulted in colitis, showing that a single protein expressed by B. theta can drive differentiation of Tregs that self-regulate Teffs to prevent disease. We found that BθOM T cells recognized a peptide derived from a single B. theta protein, BT4295, whose expression is regulated by nutrients, with glucose being a strong catabolite repressor. Mice fed a high-glucose diet had a greatly reduced activation of BθOM T cells in the colon. These studies establish that the immune response to specific bacterial antigens can be modified by changes in the diet by altering antigen expression in the microbe.

Effects of Race/Ethnicity and Socioeconomic Status on Outcome in Childhood Acute Lymphoblastic Leukemia.

With modern therapy, overall survival (OS) for children with acute lymphoblastic leukemia approaches 90%. However, inferior outcomes for minority children have been reported. Data on the effects of ethnicity/race as it relates to socioeconomic status are limited. Using state cancer registry data from Texas and Florida, we evaluated the impact of neighborhood-level poverty rate and race/ethnicity on OS for 4719 children with acute lymphoblastic leukemia. On multivariable analysis, patients residing in neighborhoods with the highest poverty rate had a 1.8-fold increase in mortality compared with patients residing in neighborhoods with the lowest poverty rate (hazard ratio [HR], 1.8; 95% confidence interval [CI], 1.41-2.30). Hispanic and non-Hispanic black patients also had increased risk of mortality compared with non-Hispanic white patients (Hispanic: HR, 1.18; 95% CI, 1.01-1.39; non-Hispanic black: HR, 1.31; 95% CI, 1.03-1.66). On subgroup analysis, there was a 21.7% difference in 5-year OS when comparing non-Hispanic white children living in the lowest poverty neighborhoods (5-year OS, 91.2%; 95% CI, 88.6-93.2) to non-Hispanic black children living in the highest poverty neighborhoods (5-year OS, 69.5%; 95% CI, 61.5-76.1). To address such disparities in survival, further work is needed to identify barriers to cancer care in this pediatric population.

Distance to Radiation Facility and Treatment Choice in Early-Stage Breast Cancer.

PURPOSE: Breast-conserving therapy (BCT) is a recommended alternative to mastectomy (MT) for early-stage breast cancer. Limited access to radiation therapy (RT) may result in higher rates of MT. We assessed the association between distance to the nearest RT facility and the use of MT, in a modern cohort of women. METHODS AND MATERIALS: Women with stage 0-II breast cancer eligible for BCT diagnosed from 2004 to 2010 were identified from the Florida Cancer Data System (FCDS). Distances from patient census tracts to the nearest RT facility census tract were calculated. Multivariate logistic regression was used to identify explanatory variables that influenced MT use. RESULTS: Of the 27,489 eligible women, 32.1% (n=8841) underwent MT, and 67.8% (n=18,648) underwent BCS. Thirty-two percent of patients lived in a census tract that was >5 miles from an RT facility. MT use increased with increasing distance to RT facility (31.1% at ≤5 miles, 33.8% at >5 to <15 miles, 34.9% at 15 to <40 miles, and 51% at ≥40 miles, P<.001). The likelihood was that MT was independently associated with increasing distance to RT facility on multivariate analysis (P<.001). Compared to patients living <5 miles away from an RT facility, patients living 15 to <40 miles away were 1.2 times more likely to be treated with MT (odds ratio [OR]: 1.19, 95% confidence interval [CI]: 1.05-1.35, P<.01), and those living ≥40 miles away were more than twice as likely to be treated with MT (OR: 2.17, 95% CI: 1.48-3.17, P<.001). However, in patients younger than 50 years (n=5179), MT use was not associated with distance to RT facility (P=.235). CONCLUSIONS: MT use in a modern cohort of women is independently associated with distance to RT facility. However, for young patients, distance to RT is not a significant explanatory variable for MT use.

Direct tests of enzymatic heme degradation by the malaria parasite Plasmodium falciparum.

Malaria parasites generate vast quantities of heme during blood stage infection via hemoglobin digestion and limited de novo biosynthesis, but it remains unclear if parasites metabolize heme for utilization or disposal. Recent in vitro experiments with a heme oxygenase (HO)-like protein from Plasmodium falciparum suggested that parasites may enzymatically degrade some heme to the canonical HO product, biliverdin (BV), or its downstream metabolite, bilirubin (BR). To directly test for BV and BR production by P. falciparum parasites, we DMSO-extracted equal numbers of infected and uninfected erythrocytes and developed a sensitive LC-MS/MS assay to quantify these tetrapyrroles. We found comparable low levels of BV and BR in both samples, suggesting the absence of HO activity in parasites. We further tested live parasites by targeted expression of a fluorescent BV-binding protein within the parasite cytosol, mitochondrion, and plant-like plastid. This probe could detect exogenously added BV but gave no signal indicative of endogenous BV production within parasites. Finally, we recombinantly expressed and tested the proposed heme degrading activity of the HO-like protein, PfHO. Although PfHO bound heme and protoporphyrin IX with modest affinity, it did not catalyze heme degradation in vivo within bacteria or in vitro in UV absorbance and HPLC assays. These observations are consistent with PfHO's lack of a heme-coordinating His residue and suggest an alternative function within parasites. We conclude that P. falciparum parasites lack a canonical HO pathway for heme degradation and thus rely fully on alternative mechanisms for heme detoxification and iron acquisition during blood stage infection.