Conjugation to Native and Nonnative Triscatecholate Siderophores Enhances Delivery and Antibacterial Activity of a ß-Lactam to Gram-Negative Bacterial Pathogens.

Developing new antibiotics and delivery strategies is of critical importance for treating infections caused by Gram-negative bacterial pathogens. Hijacking bacterial iron uptake machinery, such as that of the siderophore enterobactin (Ent), represents one promising approach toward these goals. Here, we report a novel Ent-inspired siderophore-antibiotic conjugate (SAC) employing an alternative siderophore moiety as the delivery vector and demonstrate the potency of our SACs harboring the ß-lactam antibiotic ampicillin (Amp) against multiple pathogenic Gram-negative bacterial strains. We establish the ability of N,N',N''-(nitrilotris(ethane-2,1-diyl))tris(2,3-dihydroxybenzamide) (TRENCAM, hereafter TC), a synthetic mimic of Ent, to facilitate drug delivery across the outer membrane (OM) of Gram-negative pathogens. Conjugation of Amp to a new monofunctionalized TC scaffold affords TC-Amp, which displays markedly enhanced antibacterial activity against the gastrointestinal pathogen Salmonella enterica serovar Typhimurium (STm) compared with unmodified Amp. Bacterial uptake, antibiotic susceptibility, and microscopy studies with STm show that the TC moiety facilitates TC-Amp uptake by the OM receptors FepA and IroN and that the Amp warhead inhibits penicillin-binding proteins. Moreover, TC-Amp achieves targeted activity, selectively killing STm in the presence of a commensal lactobacillus. Remarkably, we uncover that TC-Amp and its Ent-based predecessor Ent-Amp achieve enhanced antibacterial activity against diverse Gram-negative ESKAPE pathogens that express Ent uptake machinery, including strains that possess intrinsic ß-lactam resistance. TC-Amp and Ent-Amp exhibit potency comparable to that of the FDA-approved SAC cefiderocol against Gram-negative pathogens. These results demonstrate the effective application of native and appropriately designed nonnative siderophores as vectors for drug delivery across the OM of multiple Gram-negative bacterial pathogens.

GNTI-122: an autologous antigen-specific engineered Treg cell therapy for type 1 diabetes.

Tregs have the potential to establish long-term immune tolerance in patients recently diagnosed with type 1 diabetes (T1D) by preserving ß cell function. Adoptive transfer of autologous thymic Tregs, although safe, exhibited limited efficacy in previous T1D clinical trials, likely reflecting a lack of tissue specificity, limited IL-2 signaling support, and in vivo plasticity of Tregs. Here, we report a cell engineering strategy using bulk CD4+ T cells to generate a Treg cell therapy (GNTI-122) that stably expresses FOXP3, targets the pancreas and draining lymph nodes, and incorporates a chemically inducible signaling complex (CISC). GNTI-122 cells maintained an expression profile consistent with Treg phenotype and function. Activation of CISC using rapamycin mediated concentration-dependent STAT5 phosphorylation and, in concert with T cell receptor engagement, promoted cell proliferation. In response to the cognate antigen, GNTI-122 exhibited direct and bystander suppression of polyclonal, islet-specific effector T cells from patients with T1D. In an adoptive transfer mouse model of T1D, a mouse engineered-Treg analog of GNTI-122 trafficked to the pancreas, decreased the severity of insulitis, and prevented progression to diabetes. Taken together, these findings demonstrate in vitro and in vivo activity and support further development of GNTI-122 as a potential treatment for T1D.

Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets.

Foxp3+ regulatory T cells (Tregs) in the colon are key to promoting peaceful coexistence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TFs; Helios, Rorγ, Gata3, and cMaf), but their interrelationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs (Rorγ, Helios, Gata3, and cMaf) play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR repertoires in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related and cannot be uniquely equated to tTreg and pTreg. Comparison of spleen and colon repertoires revealed that 2 to 5% of clonotypes are shared between the locations. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.

Homeostatic, repertoire and transcriptional relationships between colon T regulatory cell subsets.

Foxp3 + regulatory T cells (Tregs) in the colon are key to promoting peaceful co-existence with symbiotic microbes. Differentiated in either thymic or peripheral locations, and modulated by microbes and other cellular influencers, colonic Treg subsets have been identified through key transcription factors (TF; Helios, Rorg, Gata3, cMaf), but their inter-relationships are unclear. Applying a multimodal array of immunologic, genomic, and microbiological assays, we find more overlap than expected between populations. The key TFs play different roles, some essential for subset identity, others driving functional gene signatures. Functional divergence was clearest under challenge. Single-cell genomics revealed a spectrum of phenotypes between the Helios+ and Rorγ+ poles, different Treg-inducing bacteria inducing the same Treg phenotypes to varying degrees, not distinct populations. TCR clonotypes in monocolonized mice revealed that Helios+ and Rorγ+ Tregs are related, and cannot be uniquely equated to tTreg and pTreg. We propose that rather than the origin of their differentiation, tissue-specific cues dictate the spectrum of colonic Treg phenotypes.

Regulatory T cells in the face of the intestinal microbiota.

Regulatory T cells (Treg cells) are key players in ensuring a peaceful coexistence with microorganisms and food antigens at intestinal borders. Startling new information has appeared in recent years on their diversity, the importance of the transcription factor FOXP3, how T cell receptors influence their fate and the unexpected and varied cellular partners that influence Treg cell homeostatic setpoints. We also revisit some tenets, maintained by the echo chambers of Reviews, that rest on uncertain foundations or are a subject of debate.

Treg cells require Izumo1R to regulate γδT cell-driven inflammation in the skin.

Izumo1R is a pseudo-folate receptor with an essential role in mediating tight oocyte/spermatozoa contacts during fertilization. Intriguingly, it is also expressed in CD4+ T lymphocytes, in particular Treg cells under the control of Foxp3. To understand Izumo1R function in Treg cells, we analyzed mice with Treg-specific Izumo1r deficiency (Iz1rTrKO). Treg differentiation and homeostasis were largely normal, with no overt autoimmunity and only marginal increases in PD1+ and CD44hi Treg phenotypes. pTreg differentiation was also unaffected. Iz1rTrKO mice proved uniquely susceptible to imiquimod-induced, γδT cell-dependent, skin disease, contrasting with normal responses to several inflammatory or tumor challenges, including other models of skin inflammation. Analysis of Iz1rTrKO skin revealed a subclinical inflammation that presaged IMQ-induced changes, with an imbalance of Rorγ+ γδT cells. Immunostaining of normal mouse skin revealed the expression of Izumo1, the ligand for Izumo1R, electively in dermal γδT cells. We propose that Izumo1R on Tregs enables tight contacts with γδT cells, thereby controlling a particular path of skin inflammation.

Sequestration of gut pathobionts in intraluminal casts, a mechanism to avoid dysregulated T cell activation by pathobionts.

T cells that express the transcription factor RORγ, regulatory (Treg), or conventional (Th17) are strongly influenced by intestinal symbionts. In a genetic approach to identify mechanisms underlying this influence, we performed a screen for microbial genes implicated, in germfree mice monocolonized with Escherichia coli Nissle. The loss of capsule-synthesis genes impaired clonal expansion and differentiation of intestinal RORγ+ T cells. Mechanistic exploration revealed that the capsule-less mutants remained able to induce species-specific immunoglobulin A (IgA) and were highly IgA-coated. They could still trigger myeloid cells, and more effectively damaged epithelial cells in vitro. Unlike wild-type microbes, capsule-less mutants were mostly engulfed in intraluminal casts, large agglomerates composed of myeloid cells extravasated into the gut lumen. We speculate that sequestration in luminal casts of potentially harmful microbes, favored by IgA binding, reduces the immune system's actual exposure, preserving host-microbe equilibrium. The variable immunostimulation by microbes that has been charted in recent years may not solely be conditioned by triggering molecules or metabolites but also by physical limits to immune system exposure.

Conjugation to Enterobactin and Salmochelin S4 Enhances the Antimicrobial Activity and Selectivity of ß-Lactam Antibiotics against Nontyphoidal Salmonella.

The pathogen Salmonella enterica is a leading cause of infection worldwide. Nontyphoidal Salmonella (NTS) serovars typically cause inflammatory diarrhea in healthy individuals, and can cause bacteremia in immunocompromised patients, children, and the elderly. Management of NTS infection poses a challenge because antibiotic treatment prolongs fecal shedding of the pathogen and is thus not recommended for most patients. In recent years, the emergence of antibiotic resistance in NTS has also become a major issue. Thus, new therapeutic strategies to target NTS are needed. Here, we evaluated whether six siderophore-ß-lactam conjugates based on enterobactin (Ent) and salmochelin S4 (digulcosylated Ent, DGE) provide antimicrobial activity against the two highly prevalent NTS serovars Typhimurium and Enteritidis by targeting the siderophore receptors FepA and/or IroN. The conjugates showed 10- to 1000-fold lower minimum inhibitory concentrations against both serovars Typhimurium and Enteritidis compared to the parent antibiotics under iron limitation and were recognized and transported by FepA and/or IroN. NTS treated with the Ent/DGE-ß-lactam conjugates exhibited aberrant cellular morphologies suggesting inhibition of penicillin-binding proteins, and the conjugates selectively killed NTS in coculture with Staphylococcus aureus. Lastly, the DGE-based conjugates proved to be effective at inhibiting growth of NTS in the presence of the Ent-sequestering protein lipocalin-2. This work describes the successful use of siderophore-antibiotic conjugates against NTS and highlights the opportunity for narrowing the activity spectrum of antibiotics by using Ent and DGE to target enteric bacterial pathogens.

Esterase-Catalyzed Siderophore Hydrolysis Activates an Enterobactin-Ciprofloxacin Conjugate and Confers Targeted Antibacterial Activity.

Enteric Gram-negative bacteria, including Escherichia coli, biosynthesize and deploy the triscatecholate siderophore enterobactin (Ent) in the vertebrate host to acquire iron, an essential nutrient. We report that Ent-Cipro, a synthetic siderophore-antibiotic conjugate based on the native Ent platform that harbors an alkyl linker at one of the catechols with a ciprofloxacin cargo attached, affords targeted antibacterial activity against E. coli strains that express the pathogen-associated iroA gene cluster. Attachment of the siderophore to ciprofloxacin, a DNA gyrase inhibitor and broad-spectrum antibiotic that is used to treat infections caused by E. coli, generates an inactive prodrug and guides the antibiotic into the cytoplasm of bacteria that express the Ent uptake machinery (FepABCDG). Intracellular hydrolysis of the siderophore restores the activity of the antibiotic. Remarkably, Fes, the cytoplasmic Ent hydrolase expressed by all E. coli, does not contribute to Ent-Cipro activation. Instead, this processing step requires IroD, a cytoplasmic hydrolase that is expressed only by E. coli that harbor the iroA gene cluster and are predominantly pathogenic. In the uropathogenic E. coli UTI89 and CFT073, Ent-Cipro provides antibacterial activity comparable to unmodified ciprofloxacin. This work highlights the potential of leveraging and targeting pathogen-associated microbial enzymes in narrow-spectrum antibacterial approaches. Moreover, because E. coli include harmless gut commensals as well as resident microbes that can contribute to disease, Ent-Cipro may provide a valuable chemical tool for strain-selective modulation of the microbiota.

Siderophore-based immunization strategy to inhibit growth of enteric pathogens.

Infections with Gram-negative pathogens pose a serious threat to public health. This scenario is exacerbated by increases in antibiotic resistance and the limited availability of vaccines and therapeutic tools to combat these infections. Here, we report an immunization approach that targets siderophores, which are small molecules exported by enteric Gram-negative pathogens to acquire iron, an essential nutrient, in the host. Because siderophores are nonimmunogenic, we designed and synthesized conjugates of a native siderophore and the immunogenic carrier protein cholera toxin subunit B (CTB). Mice immunized with the CTB-siderophore conjugate developed anti-siderophore antibodies in the gut mucosa, and when mice were infected with the enteric pathogen Salmonella, they exhibited reduced intestinal colonization and reduced systemic dissemination of the pathogen. Moreover, analysis of the gut microbiota revealed that reduction of Salmonella colonization in the inflamed gut was accompanied by expansion of Lactobacillus spp., which are beneficial commensal organisms that thrive in similar locales as Enterobacteriaceae. Collectively, our results demonstrate that anti-siderophore antibodies inhibit Salmonella colonization. Because siderophore-mediated iron acquisition is a virulence trait shared by many bacterial and fungal pathogens, blocking microbial iron acquisition by siderophore-based immunization or other siderophore-targeted approaches may represent a novel strategy to prevent and ameliorate a broad range of infections.

Microcins mediate competition among Enterobacteriaceae in the inflamed gut.

The Enterobacteriaceae are a family of Gram-negative bacteria that include commensal organisms as well as primary and opportunistic pathogens that are among the leading causes of morbidity and mortality worldwide. Although Enterobacteriaceae often comprise less than 1% of a healthy intestine's microbiota, some of these organisms can bloom in the inflamed gut; expansion of enterobacteria is a hallmark of microbial imbalance known as dysbiosis. Microcins are small secreted proteins that possess antimicrobial activity in vitro, but whose role in vivo has been unclear. Here we demonstrate that microcins enable the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to limit the expansion of competing Enterobacteriaceae (including pathogens and pathobionts) during intestinal inflammation. Microcin-producing EcN limits the growth of competitors in the inflamed intestine, including commensal E. coli, adherent-invasive E. coli and the related pathogen Salmonella enterica. Moreover, only therapeutic administration of the wild-type, microcin-producing EcN to mice previously infected with S. enterica substantially reduced intestinal colonization by the pathogen. Our work provides the first evidence that microcins mediate inter- and intraspecies competition among the Enterobacteriaceae in the inflamed gut. Moreover, we show that microcins can act as narrow-spectrum therapeutics to inhibit enteric pathogens and reduce enterobacterial blooms.

No vacancy: how beneficial microbes cooperate with immunity to provide colonization resistance to pathogens.

The mammalian intestine harbors a community of trillions of microbes, collectively known as the gut microbiota, which coevolved with the host in a mutually beneficial relationship. Among the numerous gut microbial species, certain commensal bacteria are known to provide health benefits to the host when administered in adequate amounts and, as such, are labeled "probiotics." We review some of the mechanisms by which probiotics and other beneficial commensals provide colonization resistance to pathogens. The battle for similar nutrients and the bacterial secretion of antimicrobials provide a direct means of competition between beneficial and harmful microbes. Beneficial microbes can also indirectly diminish pathogen colonization by stimulating the development of innate and adaptive immunity, as well as the function of the mucosal barrier. Altogether, we gather and present evidence that beneficial microbes cooperate with host immunity in an effort to shut out pathogens.

A hydrogen boost for salmonella.

Although the microbiota protects the host from infection, pathogens are still able to colonize and cause disease. In this issue of Cell Host & Microbe, Maier et al. (2013) provide mechanistic insight into the initial stages of Salmonella growth in the gut, showing that microbiota-derived hydrogen provides an energy boost to Salmonella.

Probiotics: properties, examples, and specific applications.

Probiotics are beneficial components of the microbiota that have been used for centuries because of the health benefits they confer to the host. Only recently, however, has the contribution of probiotics to modulation of immunological, respiratory, and gastrointestinal functions started to be fully appreciated and scientifically evaluated. Probiotics such as Escherichia coli Nissle 1917 and lactic acid bacteria are currently used to, or have been evaluated for use to, prevent or treat a range of intestinal maladies including inflammatory bowel disease, constipation, and colon cancer. Engineering these natural probiotics to produce immunomodulatory molecules may help to further increase the benefit to the host. In this article, we will discuss some of the mechanisms of action of probiotics as well as advances in the rational design of probiotics.