Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut.

Drug platforms that enable the directed delivery of therapeutics to sites of diseases to maximize efficacy and limit off-target effects are needed. Here, we report the development of PROT3EcT, a suite of commensal Escherichia coli engineered to secrete proteins directly into their surroundings. These bacteria consist of three modular components: a modified bacterial protein secretion system, the associated regulatable transcriptional activator, and a secreted therapeutic payload. PROT3EcT secrete functional single-domain antibodies, nanobodies (Nbs), and stably colonize and maintain an active secretion system within the intestines of mice. Furthermore, a single prophylactic dose of a variant of PROT3EcT that secretes a tumor necrosis factor-alpha (TNF-α)-neutralizing Nb is sufficient to ablate pro-inflammatory TNF levels and prevent the development of injury and inflammation in a chemically induced model of colitis. This work lays the foundation for developing PROT3EcT as a platform for the treatment of gastrointestinal-based diseases.

High-Throughput Screening of Type III Secretion Determinants Reveals a Major Chaperone-Independent Pathway.

Numerous Gram-negative bacterial pathogens utilize type III secretion systems (T3SSs) to inject tens of effector proteins directly into the cytosol of host cells. Through interactions with cognate chaperones, type III effectors are defined and recruited to the sorting platform, a cytoplasmic component of these membrane-embedded nanomachines. However, notably, a comprehensive review of the literature reveals that the secretion of most type III effectors has not yet been linked to a chaperone, raising questions regarding the existence of unknown chaperones as well as the universality of chaperones in effector secretion. Here, we describe the development of the first high-throughput type III secretion (T3S) assay, a semiautomated solid-plate-based assay, which enables the side-by-side comparison of secretion of over 20 Shigella effectors under a multitude of conditions. Strikingly, we found that the majority of Shigella effectors are secreted at equivalent levels by wild-type and variants of Shigella that no longer encode one or all known Shigella T3S effector chaperones. In addition, we found that Shigella effectors are efficiently secreted from a laboratory strain of Escherichia coli expressing the core Shigella type III secretion apparatus (T3SA) but no other Shigella-specific proteins. Furthermore, we observed that the sequences necessary and sufficient to define chaperone-dependent and -independent effectors are fundamentally different. Together, these findings support the existence of a major, previously unrecognized, noncanonical chaperone-independent secretion pathway that is likely common to many T3SSs.IMPORTANCE Many bacterial pathogens use specialized nanomachines, including type III secretion systems, to directly inject virulence proteins (effectors) into host cells. Here, we present the first extensive analysis of chaperone dependence in the process of type III effector secretion, providing strong evidence for the existence of a previously unrecognized chaperone-independent pathway. This noncanonical pathway is likely common to many bacteria, as an extensive review of the literature reveals that the secretion of multiple type III effectors has not yet been knowingly linked to a chaperone. While additional studies will be required to discern the molecular details of this pathway, its prevalence suggests that it can likely serve as a new target for the development of antimicrobial agents.

Synthetic bottom-up approach reveals the complex interplay of Shigella effectors in regulation of epithelial cell death.

Over the course of an infection, many Gram-negative bacterial pathogens use complex nanomachines to directly inject tens to hundreds of proteins (effectors) into the cytosol of infected host cells. These effectors rewire processes to promote bacterial replication and spread. The roles of effectors in pathogenesis have traditionally been investigated by screening for phenotypes associated with their absence, a top-down approach that can be limited, as effectors often act in a functionally redundant or additive manner. Here we describe a synthetic Escherichia coli-based bottom-up platform to conduct gain-of-function screens for roles of individual Shigella effectors in pathogenesis. As proof of concept, we screened for Shigella effectors that limit cell death induced on cytosolic entry of bacteria into epithelial cells. Using this platform, in addition to OspC3, an effector known to inhibit cell death via pyroptosis, we have identified OspD2 and IpaH1.4 as cell death inhibitors. In contrast to almost all type III effectors, OspD2 does not target a host cell process, but rather regulates the activity of the Shigella type III secretion apparatus limiting the cytosolic delivery (translocation) of effectors during an infection. Remarkably, by limiting the translocation of a single effector, VirA, OspD2 controls the timing of epithelial cell death via calpain-mediated necrosis. Together, these studies provide insight into the intricate manner by which Shigella effectors interact to establish a productive intracytoplasmic replication niche before the death of infected epithelial cells.

QseC inhibition as an antivirulence approach for colitis-associated bacteria.

Hosts and their microbes have established a sophisticated communication system over many millennia. Within mammalian hosts, this dynamic cross-talk is essential for maintaining intestinal homeostasis. In a genetically susceptible host, dysbiosis of the gut microbiome and dysregulated immune responses are central to the development of inflammatory bowel disease (IBD). Previous surveys of stool from the T-bet-/-Rag2-/- IBD mouse model revealed microbial features that discriminate between health and disease states. Enterobacteriaceae expansion and increased gene abundances for benzoate degradation, two-component systems, and bacterial motility proteins pointed to the potential involvement of a catecholamine-mediated bacterial signaling axis in colitis pathogenesis. Enterobacteriaceae sense and respond to microbiota-generated signals and host-derived catecholamines through the two-component quorum-sensing Escherichia coli regulators B and C (QseBC) system. On signal detection, QseC activates a cascade to induce virulence gene expression. Although a single pathogen has not been identified as a causative agent in IBD, adherent-invasive Escherichia coli (AIEC) have been implicated. Flagellar expression is necessary for the IBD-associated AIEC strain LF82 to establish colonization. Thus, we hypothesized that qseC inactivation could reduce LF82's virulence, and found that an absence of qseC leads to down-regulated flagellar expression and motility in vitro and reduced colonization in vivo. We extend these findings on the potential of QseC-based IBD therapeutics to three preclinical IBD models, wherein we observe that QseC blockade can effectively modulate colitogenic microbiotas to reduce intestinal inflammation. Collectively, our data support a role for QseC-mediated bacterial signaling in IBD pathogenesis and indicate that QseC inhibition may be a useful microbiota-targeted approach for disease management.

The type III secretion system apparatus determines the intracellular niche of bacterial pathogens.

Upon entry into host cells, intracellular bacterial pathogens establish a variety of replicative niches. Although some remodel phagosomes, others rapidly escape into the cytosol of infected cells. Little is currently known regarding how professional intracytoplasmic pathogens, including Shigella, mediate phagosomal escape. Shigella, like many other Gram-negative bacterial pathogens, uses a type III secretion system to deliver multiple proteins, referred to as effectors, into host cells. Here, using an innovative reductionist-based approach, we demonstrate that the introduction of a functional Shigella type III secretion system, but none of its effectors, into a laboratory strain of Escherichia coli is sufficient to promote the efficient vacuole lysis and escape of the modified bacteria into the cytosol of epithelial cells. This establishes for the first time, to our knowledge, a direct physiologic role for the Shigella type III secretion apparatus (T3SA) in mediating phagosomal escape. Furthermore, although protein components of the T3SA share a moderate degree of structural and functional conservation across bacterial species, we show that vacuole lysis is not a common feature of T3SA, as an effectorless strain of Yersinia remains confined to phagosomes. Additionally, by exploiting the functional interchangeability of the translocator components of the T3SA of Shigella, Salmonella, and Chromobacterium, we demonstrate that a single protein component of the T3SA translocon-Shigella IpaC, Salmonella SipC, or Chromobacterium CipC-determines the fate of intracellular pathogens within both epithelial cells and macrophages. Thus, these findings have identified a likely paradigm by which the replicative niche of many intracellular bacterial pathogens is established.

Transfer of Large Contiguous DNA Fragments onto a Low Copy Plasmid or into the Bacterial Chromosome.

Bacterial pathogenicity islands and other contiguous operons can be difficult to clone using conventional methods due to their large size. Here we describe a robust 3-step method to transfer large defined fragments of DNA from virulence plasmids or cosmids onto smaller autonomously replicating plasmids or directly into defined sites in the bacterial chromosome that incorporates endogenous yeast and λ Red homologous recombination systems. This methodology has been successfully used to isolate and integrate at least 31 kb of contiguous DNA and can be readily adapted for the recombineering of E. coli and its close relatives.

Engineering Escherichia coli into a protein delivery system for mammalian cells.

Many Gram-negative pathogens encode type 3 secretion systems, sophisticated nanomachines that deliver proteins directly into the cytoplasm of mammalian cells. These systems present attractive opportunities for therapeutic protein delivery applications; however, their utility has been limited by their inherent pathogenicity. Here, we report the reengineering of a laboratory strain of Escherichia coli with a tunable type 3 secretion system that can efficiently deliver heterologous proteins into mammalian cells, thereby circumventing the need for virulence attenuation. We first introduced a 31 kB region of Shigella flexneri DNA that encodes all of the information needed to form the secretion nanomachine onto a plasmid that can be directly propagated within E. coli or integrated into the E. coli chromosome. To provide flexible control over type 3 secretion and protein delivery, we generated plasmids expressing master regulators of the type 3 system from either constitutive or inducible promoters. We then constructed a Gateway-compatible plasmid library of type 3 secretion sequences to enable rapid screening and identification of sequences that do not perturb function when fused to heterologous protein substrates and optimized their delivery into mammalian cells. Combining these elements, we found that coordinated expression of the type 3 secretion system and modified target protein substrates produces a nonpathogenic strain that expresses, secretes, and delivers heterologous proteins into mammalian cells. This reengineered system thus provides a highly flexible protein delivery platform with potential for future therapeutic applications.

Monocarbonyl analogs of curcumin inhibit growth of antibiotic sensitive and resistant strains of Mycobacterium tuberculosis.

Tuberculosis (TB) is a major public health concern worldwide with over 2 billion people currently infected. The rise of strains of Mycobacterium tuberculosis (Mtb) that are resistant to some or all first and second line antibiotics, including multidrug-resistant (MDR), extensively drug resistant (XDR) and totally drug resistant (TDR) strains, is of particular concern and new anti-TB drugs are urgently needed. Curcumin, a natural product used in traditional medicine in India, exhibits anti-microbial activity that includes Mtb, however it is relatively unstable and suffers from poor bioavailability. To improve activity and bioavailability, mono-carbonyl analogs of curcumin were synthesized and screened for their capacity to inhibit the growth of Mtb and the related Mycobacterium marinum (Mm). Using disk diffusion and liquid culture assays, we found several analogs that inhibit in vitro growth of Mm and Mtb, including rifampicin-resistant strains. Structure activity analysis of the analogs indicated that Michael acceptor properties are critical for inhibitory activity. However, no synergistic effects were evident between the monocarbonyl analogs and rifampicin on inhibiting growth. Together, these data provide a structural basis for the development of analogs of curcumin with pronounced anti-mycobacterial activity and provide a roadmap to develop additional structural analogs that exhibit more favorable interactions with other anti-TB drugs.

Disparities in capreomycin resistance levels associated with the rrs A1401G mutation in clinical isolates of Mycobacterium tuberculosis.

As the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. The rrs A1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between the rrs A1401G mutation and CAP resistance, with up to 40% of rrs A1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring the rrs A1401G mutation and found that the CAP MICs ranged from 8 µg/ml to 40 µg/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 µg/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 µg/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.

Aminoglycoside cross-resistance in Mycobacterium tuberculosis due to mutations in the 5' untranslated region of whiB7.

Since the discovery of streptomycin's bactericidal activity against Mycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5' untranslated region of the transcriptional activator whiB7 that confer low-level resistance to both aminoglycosides. The mutations lead to 23- to 145-fold increases in whiB7 transcripts and subsequent increased expression of both eis (Rv2416c) and tap (Rv1258c). Increased expression of eis confers kanamycin resistance in these mutants, while increased expression of tap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency in whiB7 mutants than in a wild-type (WT) strain. Although whiB7 is typically associated with intrinsic antibiotic resistance in M. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region of whiB7 contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.