Combined deletion of cathepsin protease family members reveals compensatory mechanisms in cancer.

Proteases are important for regulating multiple tumorigenic processes, including angiogenesis, tumor growth, and invasion. Elevated protease expression is associated with poor patient prognosis across numerous tumor types. Several multigene protease families have been implicated in cancer, including cysteine cathepsins. However, whether individual family members have unique roles or are functionally redundant remains poorly understood. Here we demonstrate stage-dependent effects of simultaneously deleting cathepsin B (CtsB) and CtsS in a murine pancreatic neuroendocrine tumor model. Early in tumorigenesis, the double knockout results in an additive reduction in angiogenic switching, whereas at late stages, several tumorigenic phenotypes are unexpectedly restored to wild-type levels. We identified CtsZ, which is predominantly supplied by tumor-associated macrophages, as the compensatory protease that regulates the acquired tumor-promoting functions of lesions deficient in both CtsB and CtsS. Thus, deletion of multiple cathepsins can lead to stage-dependent, compensatory mechanisms in the tumor microenvironment, which has potential implications for the clinical consideration of selective versus pan-family cathepsin inhibitors in cancer.

Distinct functions of macrophage-derived and cancer cell-derived cathepsin Z combine to promote tumor malignancy via interactions with the extracellular matrix.

During the process of tumor progression, cancer cells can produce the requisite growth- and invasion-promoting factors and can also rely on noncancerous cells in the tumor microenvironment as an alternative, cell-extrinsic source. However, whether the cellular source influences the function of such tumor-promoting factors remains an open question. Here, we examined the roles of the cathepsin Z (CtsZ) protease, which is provided by both cancer cells and macrophages in pancreatic neuroendocrine tumors in humans and mice. We found that tumor proliferation was exclusively regulated by cancer cell-intrinsic functions of CtsZ, whereas tumor invasion required contributions from both macrophages and cancer cells. Interestingly, several of the tumor-promoting functions of CtsZ were not dependent on its described catalytic activity but instead were mediated via the Arg-Gly-Asp (RGD) motif in the enzyme prodomain, which regulated interactions with integrins and the extracellular matrix. Together, these results underscore the complexity of interactions within the tumor microenvironment and indicate that cellular source can indeed impact molecular function.

ClpX Is Essential and Activated by Single-Strand DNA Binding Protein in Mycobacteria.

The ClpP1P2 proteolytic complex is essential in Mycobacterium tuberculosis Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of clpX transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria.IMPORTANCE Tuberculosis, caused by Mycobacterium tuberculosis, imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.

Fluorescence Imaging-Based Discovery of Membrane Domain-Associated Proteins in Mycobacterium smegmatis.

Mycobacteria spatially organize their plasma membrane, and many enzymes involved in envelope biosynthesis associate with a membrane compartment termed the intracellular membrane domain (IMD). The IMD is concentrated in the polar regions of growing cells and becomes less polarized under nongrowing conditions. Because mycobacteria elongate from the poles, the observed polar localization of the IMD during growth likely supports the localized biosynthesis of envelope components. While we have identified more than 300 IMD-associated proteins by proteomic analyses, only a few of these have been verified by independent experimental methods. Furthermore, some IMD-associated proteins may have escaped proteomic identification and remain to be identified. Here, we visually screened an arrayed library of 523 Mycobacterium smegmatis strains, each producing a Dendra2-FLAG-tagged recombinant protein. We identified 29 fusion proteins that showed polar fluorescence patterns characteristic of IMD proteins. Twenty of these had previously been suggested to localize to the IMD based on proteomic data. Of the nine remaining IMD candidate proteins, three were confirmed by biochemical methods to be associated with the IMD. Taken together, this new colocalization strategy is effective in verifying the IMD association of proteins found by proteomic analyses while facilitating the discovery of additional IMD-associated proteins. IMPORTANCE The intracellular membrane domain (IMD) is a membrane subcompartment found in Mycobacterium smegmatis cells. Proteomic analysis of purified IMD identified more than 300 proteins, including enzymes involved in cell envelope biosynthesis. However, proteomics on its own is unlikely to detect every IMD-associated protein because of technical and biological limitations. Here, we describe fluorescent protein colocalization as an alternative, independent approach. Using a combination of fluorescence microscopy, proteomics, and subcellular fractionation, we identified three new proteins associated with the IMD. Such a robust method to rigorously define IMD proteins will benefit future investigations to decipher the synthesis, maintenance, and functions of this membrane domain and help delineate a more general mechanism of subcellular protein localization in mycobacteria.

Application of a gut-immune co-culture system for the study of N-glycan-dependent host-pathogen interactions of Campylobacter jejuni.

An in vitro gut-immune co-culture model with apical and basal accessibility, designed to more closely resemble a human intestinal microenvironment, was employed to study the role of the N-linked protein glycosylation pathway in Campylobacter jejuni pathogenicity. The gut-immune co-culture (GIC) was developed to model important aspects of the human small intestine by the inclusion of mucin-producing goblet cells, human enterocytes and dendritic cells, bringing together a mucus-containing epithelial monolayer with elements of the innate immune system. The utility of the system was demonstrated by characterizing host-pathogen interactions facilitated by N-linked glycosylation, such as host epithelial barrier functions, bacterial invasion and immunogenicity. Changes in human intestinal barrier functions in the presence of 11168 C. jejuni (wildtype) strains were quantified using GICs. The glycosylation-impaired strain 11168 ΔpglE was 100-fold less capable of adhering to and invading this intestinal model in cell infectivity assays. Quantification of inflammatory signaling revealed that 11168ΔpglE differentially modulated inflammatory responses in different intestinal microenvironments, suppressive in some but activating in others. Virulence-associated outer membrane vesicles produced by wildtype and 11168ΔpglE C. jejuni were shown to have differential composition and function, with both leading to immune system activation when provided to the gut-immune co-culture model. This analysis of aspects of C. jejuni infectivity in the presence and absence of its N-linked glycome is enabled by application of the gut-immune model, and we anticipate that this system will be applicable to further studies of C. jejuni and other enteropathogens of interest.

Clostridioides difficile-Associated Antibiotics Alter Human Mucosal Barrier Functions by Microbiome-Independent Mechanisms.

A clinically relevant risk factor for Clostridioides difficile-associated disease (CDAD) is recent antibiotic treatment. Although broad-spectrum antibiotics have been shown to disrupt the structure of the gut microbiota, some antibiotics appear to increase CDAD risk without being highly active against intestinal anaerobes, suggesting direct nonantimicrobial effects. We examined cell biological effects of antibiotic exposure that may be involved in bacterial pathogenesis using an in vitro germfree human colon epithelial culture model. We found a marked loss of mucosal barrier and immune function with exposure to the CDAD-associated antibiotics clindamycin and ciprofloxacin, distinct from the results of pretreatment with an antibiotic unassociated with CDAD, tigecycline, which did not reduce innate immune or mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibiotics sensitized mucosal barriers to C. difficile toxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent gut mucosal barrier changes in the increased risk of CDAD with specific antibiotics and warrant further studies in in vivo systems. We anticipate this work to suggest potential avenues of research for host-directed treatment and preventive therapies for CDAD.

Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer.

The microenvironment is known to critically modulate tumor progression, yet its role in regulating treatment response is poorly understood. Here we found increased macrophage infiltration and cathepsin protease levels in mammary tumors following paclitaxel (Taxol) chemotherapy. Cathepsin-expressing macrophages protected against Taxol-induced tumor cell death in coculture, an effect fully reversed by cathepsin inhibition and mediated partially by cathepsins B and S. Macrophages were also found to protect against tumor cell death induced by additional chemotherapeutics, specifically etoposide and doxorubicin. Combining Taxol with cathepsin inhibition in vivo significantly enhanced efficacy against primary and metastatic tumors, supporting the therapeutic relevance of this effect. Additionally incorporating continuous low-dose cyclophosphamide dramatically impaired tumor growth and metastasis and improved survival. This study highlights the importance of integrated targeting of the tumor and its microenvironment and implicates macrophages and cathepsins in blunting chemotherapeutic response.

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria.

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1's catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress.

Persisters and beyond: mechanisms of phenotypic drug resistance and drug tolerance in bacteria.

One of the challenges in clinical infectious diseases is the problem of chronic infections, which can require long durations of antibiotic treatment and often recur. An emerging explanation for the refractoriness of some infections to treatment is the existence of subpopulations of drug tolerant cells. While typically discussed as "persister" cells, it is becoming increasingly clear that there is significant heterogeneity in drug responses within a bacterial population and that multiple mechanisms underlie the emergence of drug tolerant and drug-resistant subpopulations. Many of these parallel mechanisms have been shown to affect drug susceptibility at the level of a whole population. Here we review mechanisms of phenotypic drug tolerance and resistance in bacteria with the goal of providing a framework for understanding the similarities and differences in these cells.

Spatiotemporal localization of proteins in mycobacteria.

Although prokaryotic organisms lack traditional organelles, they must still organize cellular structures in space and time, challenges that different species solve differently. To systematically define the subcellular architecture of mycobacteria, we perform high-throughput imaging of a library of fluorescently tagged proteins expressed in Mycobacterium smegmatis and develop a customized computational pipeline, MOMIA and GEMATRIA, to analyze these data. Our results establish a spatial organization network of over 700 conserved mycobacterial proteins and reveal a coherent localization pattern for many proteins of known function, including those in translation, energy metabolism, cell growth and division, as well as proteins of unknown function. Furthermore, our pipeline exploits morphologic proxies to enable a pseudo-temporal approximation of protein localization and identifies previously uncharacterized cell-cycle-dependent dynamics of essential mycobacterial proteins. Collectively, these data provide a systems perspective on the subcellular organization of mycobacteria and provide tools for the analysis of bacteria with non-standard growth characteristics.

Primary human colonic mucosal barrier crosstalk with super oxygen-sensitive Faecalibacterium prausnitzii in continuous culture.

BACKGROUND: The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. METHODS: Here, we investigated the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a Gut-MIcrobiome (GuMI) physiome platform that we designed and fabricated. FINDINGS: Long-term continuous co-culture of F. prausnitzii for two days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, resulted in a strictly anaerobic apical environment fostering growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identified elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrated anti-inflammatory effects of F. prausnitzii through HDAC and the TLR-NFKB axis. Finally, we identified that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. CONCLUSIONS: Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies employing this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria.