The strategic logic behind targeting secreted proteins is compelling. Many classic tuberculosis drugs must cross the bacterium’s notoriously robust and impermeable cell wall to reach their cytosolic targets, and those that do make it inside can still be neutralized by efflux pumps or other xenobiotic defenses. Secreted proteins, by contrast, sit outside or on the surface of this barrier, at the interface between pathogen and host. A drug acting on them does not necessarily need to penetrate the cell wall at all, which may in principle bypass a whole class of resistance mechanisms. Moreover, disabling virulence factors rather than killing the bacterium outright could reduce the selective pressure that drives antibiotic resistance, a persistent threat in tuberculosis therapy, where current multi-drug regimens must be taken for months and adherence failures fuel drug-resistant strains.

The review begins with the machinery itself. M. tuberculosis exports its proteins through three principal systems: the ESX, or Type VII, secretion systems, of which there are five, designated ESX-1 through ESX-5; the highly conserved general secretory, or Sec, pathway; and the twin-arginine translocation, or Tat, pathway. ESX-1 is the best characterized and secretes a stable heterodimer of EsxA, also known as the 6-kDa early secretory antigenic target ESAT-6, and EsxB, or CFP-10. The ESX-1 system is encoded in the region of difference 1 locus, which is absent from all strains of the BCG vaccine, and its loss is thought to have contributed to the attenuation of the vaccine strain, underscoring just how central this system is to virulence. The Sec pathway uses ATPases SecA1 and SecA2 with the SecYEG channel to export unfolded proteins, while the Tat pathway moves folded proteins through TatA, TatB, and TatC. ESX-3 and ESX-5 have also been implicated in virulence, whereas ESX-2 and ESX-4 remain poorly understood.

EsxA and EsxB top the list of druggable virulence factors. EsxA, a small 6-kDa helical protein, dissociates from its 10-kDa partner under acidic conditions and forms membrane-permeabilizing structures that rupture the phagosome, allowing the bacterium to escape into the host cell’s cytosol. EsxA also dampens pro-inflammatory cytokine production by binding toll-like receptor 2 and promotes apoptosis by upregulating caspases. EsxB is less understood but stabilizes EsxA and interacts with the EccCb1 ATPase of the secretion apparatus. Inhibiting this axis has required creative chemistry because protein-protein interactions are notoriously difficult to disrupt with conventional small molecules. Nevertheless, proof-of-concept exists: the small molecule IMB-BZ blocks EsxB’s interaction with EccCb1 and, in Mycobacterium marinum infection models, reduced bacterial cytolytic activity to levels comparable to an ESX-1-deficient strain while rescuing phagosome maturation. HCL2, a peptide derived from human cytochrome c oxidase subunit 3, binds EsxA and disrupts the EsxA-EsxB complex, and an antibody-derived ESAT-6-specific nanobody expressed in the cytoplasm of host cells has blocked M. tuberculosis growth in macrophages. Indirect approaches are also viable: ethoxzolamide, an FDA-approved carbonic anhydrase inhibitor, downregulates the PhoPR regulon and suppresses ESX-1 secretion, producing a phenotype resembling the attenuated PhoPR mutant in macrophages and mice.

The antigen 85 complex represents a more enzymatically tractable target. Comprising the homologous proteins Ag85A, Ag85B, and Ag85C, secreted via the Sec pathway in a roughly 2:3:1 ratio, this complex acts as a mycolyl-transferase that attaches mycolic acids to arabinogalactan and catalyzes the biosynthesis of trehalose dimycolate, the cord factor critical for the bacterium’s cord-like appearance and survival. The complex also binds fibronectin, facilitating host cell attachment and invasion. Inhibitor development began with trehalose analogues that mimicked natural substrates, but these showed limited potency and whole-cell activity. More promising are ebselen and its derivatives, which allosterically inhibit the enzyme through covalent modification of Cys209 on Ag85C, and cyclophostin and cyclipostin analogues that covalently target the catalytic Ser124. The cyclipostin analogues have inhibited intracellular growth of M. tuberculosis with limited host-cell cytotoxicity, while ebselen’s activity in macrophage models still requires validation. Notably, ebselen has been shown to potentiate the activity of streptomycin, suggesting cell-envelope targeting can synergize with existing antibiotics. Functional redundancy among the three isoforms is a challenge, but their highly conserved active sites offer a rationale for multi-isoform inhibitors that raise the mutational threshold for resistance.

Among the phosphatases, SapM, PtpA, and PtpB each sabotage the phagosome in distinct ways. SapM, a 28-kDa lipid phosphatase, strips phosphatidylinositol 3-phosphate from the phagosomal membrane, halting phagosome maturation and phagosome-lysosome fusion; mutants lacking SapM fail to arrest maturation and grow poorly in macrophages and guinea pig tissues. Competitive inhibitor 2-phospho-L-ascorbic acid showed limited potency, but newer uncompetitive inhibitors, including Tyrphostin 51 and Tyrphostin Ag183 with IC50 values of 6.3 and 8.2 micromolar respectively, sustain inhibition even at high substrate concentrations. PtpA dephosphorylates host VPS33B and excludes the vacuolar H+-ATPase to block acidification, while also suppressing JNK and p38 MAPK signaling. Unfortunately, PtpA closely resembles human protein tyrosine phosphatases such as PTP1B, so most reported inhibitors, from stevastelins to prodigiosins and chalcones, lack specificity, though some low-micromolar chalcones did inhibit intracellular growth. PtpB, by contrast, has low similarity to human PTP1B and is therefore a more attractive target; inhibitors including I-A09, L01Z08, Kuwanon G, and Fusarielin M have all reduced bacterial burden in infected macrophages, and phosphatase inhibitors have been shown to augment the bactericidal activity of the standard drug regimen.

Protein kinase G, or PknG, presents a different risk profile. This 82-kDa serine/threonine kinase blocks phagosome-lysosome fusion and promotes survival under hypoxic stress, but it shares high homology with eukaryotic kinases, raising the specter of off-target effects on host signal transduction. The landmark inhibitor AX20017, however, achieved high selectivity for mycobacterial PknG over 28 archetypal human kinases, thanks to unique amino acid side chains within its narrow binding pocket, and restored lysosomal localization of bacteria in a dose-dependent manner. Later inhibitors, including AZD7762, R406, NU-6027, sclerotiorin, RO9021, and natural flavanones, showed activity against intracellular mycobacteria, though AZD7762’s mild macrophage cytotoxicity and the unvalidated efficacy of the newest compounds illustrate how selective this field still is. The toxin channel CpnT, whose C-terminal tuberculosis necrotizing toxin hydrolyzes NAD+ to kill macrophages, and the acyl carrier protein AcpM, which suppresses apoptosis, reactive oxygen species, and autophagy while also serving fatty acid biosynthesis, currently have no direct inhibitors reported, though the endogenous antitoxin IFT offers a template for future molecules.

Lipoproteins and heat shock proteins round out the review. Lipoproteins such as LprA and LpqH act as TLR2 agonists driving TNF and IL-12 production, while LprG, LppX, and LpqN support lipid transport and cell wall homeostasis; because they are non-redundant, immunogenic, and often essential for intracellular survival, disrupting their maturation or export could simultaneously compromise bacterial viability and restore immune clearance. Among the heat shock proteins, HspX, part of the DosR dormancy regulon, drives immunosuppressive M2 macrophage polarization, and a human monoclonal IgA antibody called 2E9 against HspX has reduced lung bacterial load through enhanced phagocytosis. Hsp70, a key chaperone maintaining proteostasis during oxidative stress, hypoxia, and nutrient deprivation, is targeted by telaprevir, a repurposed inhibitor that enhanced aminoglycoside efficacy and reduced rifampicin resistance, hinting that destabilizing stress-response networks could synergize bactericidally with existing drugs.

The authors are candid about the obstacles. Some secreted proteins are also immunodominant antigens, so inhibiting them risks compromising protective host immune responses, and standard growth-inhibition screens are poorly suited to targets whose main contribution is virulence rather than in vitro growth. They propose prioritizing dual-function targets like the antigen 85 complex, which is both an immunodominant T-cell antigen and essential for cord factor biosynthesis, and point to CRISPRi-mediated gene silencing in Mycobacterium abscessus as a model for validating target vulnerability during infection. With multiple new drugs now advancing through the tuberculosis pipeline and clinical trials testing entirely new regimens, the authors argue this is an opportune moment to develop anti-virulence agents as adjuncts to standard chemotherapy. If potency, selectivity, and delivery challenges can be overcome, the proteins M. tuberculosis painstakingly exports to disarm its host may become the very handle by which modern medicine finally pulls the pathogen out of its hiding places.

Subject of Research: Secreted virulence proteins of Mycobacterium tuberculosis as therapeutic targets in tuberculosis drug discovery

Article Title: Secreted proteins of Mycobacterium tuberculosis as therapeutic targets for drug discovery

Article References: Kaweesa, A. M., & Tiwari, D. (2026). Secreted proteins of Mycobacterium tuberculosis as therapeutic targets for drug discovery. Frontiers in Tuberculosis, 4, Article 1736139. https://doi.org/10.3389/ftubr.2026.1736139

Image Credits: AI Generated

DOI: 10.3389/ftubr.2026.1736139

Keywords: tuberculosis, Mycobacterium tuberculosis, secreted proteins, ESX-1, ESAT-6, antigen 85, virulence factors, drug discovery, phosphatases, protein kinase G, heat shock proteins, antimicrobial resistance

Bacteroides fragilis is one of the most abundant bacterial species colonizing the human colon, and its relationship with its host is famously double-edged. Certain strains carry a toxin gene and are strongly associated with promoting colorectal tumors, producing the so-called Bacteroides fragilis toxin that damages DNA and drives inflammation. Yet other strains, lacking this enterotoxin, have been linked in previous studies to beneficial or protective effects, including the suppression of tumor growth in experimental models. The Chinese team worked with a human-derived non-enterotoxigenic strain precisely because of this paradox: if the same species can both promote and restrain cancer, then the specific molecules each strain produces become the decisive variable. Identifying those molecules is the first step toward understanding the mechanism and, eventually, toward developing microbiota-based therapies.

The methodological heart of the study is an approach known as activity-guided fractionation, a strategy long used in natural product chemistry to find the active needle in a haystack of molecules. The researchers first grew their bacterial strain in liquid culture and collected the conditioned supernatant, the cell-free broth containing everything the bacterium had secreted into its environment. They then tested this crude material against HT-29 cells, a well-established human colorectal cancer cell line, to confirm that the supernatant indeed inhibited cell proliferation. With that baseline activity established, they began to chop the complex mixture into progressively simpler fractions, testing each one for anti-proliferative activity along the way. Only fractions that retained activity were carried forward, ensuring that the analytical effort concentrated on the parts of the mixture most likely to contain the bioactive molecules.

The fractionation pipeline was deliberately multi-stage. Ultrafiltration first separated the supernatant by molecular weight, dividing proteins and other large molecules from smaller metabolites. Solvent extraction then partitioned the material according to chemical properties, distinguishing hydrophilic components from those that dissolve in organic solvents. Finally, chromatography provided a finer resolution, separating molecules on the basis of their interactions with a stationary phase. At each step, the researchers returned to the HT-29 cell assay, tracking where the anti-proliferative activity went. This iterative process, in which biological activity is used as a compass for chemical purification, is what distinguishes the study from a purely descriptive catalog of bacterial proteins. It converts an overwhelming proteomic inventory into a focused set of suspects.

Once the active fractions had been identified, the team turned to liquid chromatography coupled with tandem mass spectrometry, or LC-MS/MS, to determine which proteins were present in each. In this technique, proteins are digested into peptides, separated by liquid chromatography, and then fragmented inside the mass spectrometer; the resulting fragmentation patterns allow researchers to infer the identity and sequence of the original proteins with high confidence. Comparative proteomic analysis across the active and inactive fractions revealed 41 candidate proteins consistently associated with the bioactive material. The comparison is the critical element: proteins found in active fractions but absent from inactive ones, or enriched in the former, become candidates, while proteins present everywhere are unlikely to explain the specific biological effect.

Functional annotation of these 41 candidates revealed striking patterns. Many of the proteins are involved in iron acquisition, a vital survival function for bacteria living in the iron-restricted environment of the gut, where hosts actively withhold this metal from microbes. Others belong to ATP-binding cassette transport systems, molecular pumps that use the energy of ATP hydrolysis to move substrates across bacterial membranes, and to protein secretion pathways, the machinery by which bacteria export molecules into their surroundings. The enrichment of these functional categories suggests that the bioactivity may be tied to the bacterium’s interactions with its environment, including its host, rather than to housekeeping functions inside the cell.

Perhaps the most intriguing bioinformatic finding concerns protein localization. A substantial proportion of the 41 candidates were predicted to reside in the bacterial outer membrane or to carry signal peptides, the short amino acid tags that direct proteins out of the cell. This distribution raises the possibility that some of the candidate proteins travel in or on outer membrane vesicles, small spherical blebs that Gram-negative bacteria such as Bacteroides release into their environment. Outer membrane vesicles have attracted intense interest in microbiome research because they can package proteins, lipids, and genetic material and deliver them to host cells, potentially modulating immune responses and signaling pathways. If the anti-proliferative molecules are indeed shuttled in vesicles, that would offer a plausible route by which a gut bacterium could influence the behavior of epithelial cells lining the colon.

The authors are careful to frame their findings as a candidate list rather than a proof of mechanism, and this caution is scientifically appropriate. Correlation between the presence of a protein in an active fraction and the fraction’s biological effect does not establish causation. Any of the 41 proteins, or some combination of them, could be responsible for the anti-proliferative activity, or the true effector could be a non-protein molecule that co-purified with them. The researchers explicitly state that the candidates should be regarded as associated with the bioactive fractions rather than confirmed functional effectors, and that the individual and combined roles of these proteins require direct experimental validation. Such validation would typically involve purifying each candidate, testing it alone and in combination on cancer cells, and knocking out or silencing the corresponding genes in the bacterium to see whether activity disappears.

The broader significance of the study lies in the growing recognition that gut microbiota-derived factors contribute to colorectal cancer progression and, potentially, to its prevention. Epidemiological and experimental work over the past decade has implicated bacterial metabolites, toxins, and cell surface components in virtually every stage of tumor development, from DNA damage to immune evasion. Yet the field has struggled with a fundamental problem: the gut contains hundreds of bacterial species producing thousands of molecules, and pinpointing which of them matter for a given disease outcome is enormously difficult. Studies like this one demonstrate a tractable path forward. By coupling a reductionist fractionation workflow with unbiased proteomics and rigorous bioinformatics, researchers can convert a vague observation, that a bacterium seems to influence cancer cells, into a concrete, testable molecular hypothesis.

Looking ahead, the framework established by the Zunyi Medical University team could be applied to other bacterial strains and other cancer models, and the 41 candidates themselves provide a rich starting point for mechanistic work. If one or more of these proteins is eventually confirmed to suppress colorectal cancer cell proliferation, it could inspire entirely new classes of therapeutics derived from the microbiome, whether as purified proteins, engineered bacterial strains, or vesicle-based delivery systems. Conversely, even negative results from follow-up validation would sharpen the field’s understanding of how bioactive fractions exert their effects. For now, the study stands as a careful, methodical piece of science: a reminder that the answers to some of the most pressing questions in cancer biology may be produced by organisms already living inside us, waiting to be identified one protein at a time.

Subject of Research: Proteomic identification of secreted Bacteroides fragilis proteins associated with anti-colorectal cancer activity

Article Title: Proteomic identification of secreted protein candidates mediating anti-colorectal cancer activity in a human-derived non-enterotoxigenic Bacteroides fragilis

Article References: Peng, Y., Huang, L., Chen, M., Yang, H., Gan, Y., Wang, N., Wang, M., Yang, Z., & Li, J. (2026). Proteomic identification of secreted protein candidates mediating anti-colorectal cancer activity in a human-derived non-enterotoxigenic Bacteroides fragilis. BMC Cancer. https://doi.org/10.1186/s12885-026-17063-2

Image Credits: AI Generated

DOI: 10.1186/s12885-026-17063-2

Keywords: Bacteroides fragilis, colorectal cancer, proteomics, microbiome, activity-guided fractionation, LC-MS/MS, outer membrane vesicles, HT-29 cells, secreted proteins, ABC transporters, iron acquisition, gut microbiota