Michael Niederweis

Bacteriophages are highly specific predators of bacterial hosts, but the molecular mechanisms governing their entry across the complex mycobacterial cell envelope remain poorly defined. Utilizing a genome-wide CRISPR interference library in the model organism Mycobacterium smegmatis against a phylogenetically representative collection of 70 mycobacteriophages, and subsequent efficiency of plating assays, we mapped the host susceptibility determinants for phage infection. This screen established a spatial continuum of infection requirements spanning from the surface accessible outer membrane components down to cytoplasmic proteins. The four major outer membrane Mycobacterium smegmatis porins (Msp) emerged as the dominant candidate receptor family for 46 diverse phages, representing the first protein receptors proposed within this group. Interrogating the asymmetric outer bilayer also implicated cell envelope glycolipids as candidate receptors for 14 phages, resolving into glycopeptidolipid sub-lineages. Beneath the M. smegmatis mycomembrane, arabinogalactan functions as a determinant for 5 phages, while the inner membrane lipid transporter MmpL11 and the global nucleoid-associated protein Lsr2 represent determinant factors for 14 and 31 phages respectively. Although physical deletions of major porins genes trigger spontaneous regulatory pathways that upregulate silent backup paralogues, transcriptional knockdowns circumvent suppressor accumulation, revealing single-gene dependencies. Cluster analysis demonstrated that host determinant usage decouples from whole-genome viral phylogeny, indicating that standard phylogenetic groupings fail to predict surface recognition profiles. Ultimately, mapping these host dependencies reveals an evolutionary strategy where phages target highly redundant or abundant host proteins to exploit physiological liabilities, presenting a robust molecular blueprint for the rational engineering of escape-resistant therapeutic phage cocktails.

Schitoviruses are widespread prokaryotic viruses that encapsidate a giant ( ~ 3500-residue) virion-associated RNA polymerase (vRNAP). During infection, vRNAP is expelled into Gram-negative bacteria, along with two additional ejection proteins, to assemble a transient DNA-ejectosome that becomes transcriptionally active, initiating viral replication. Here, we present an integrative structural analysis of the coliphage N4 vRNAP (gp50). We find that this 383 kDa enzyme is a multi-domain, single-chain RNA polymerase, structurally distinct from both compact single-chain RNAPs and large multi-subunit holoenzymes. vRNAP is composed of loosely connected domains and exhibits an intramolecular mode of allosteric regulation through its C-terminal domain. Comparative analysis of intact and genome-released virions identified gp51, which forms an outer-membrane complex, and gp52, which assembles a periplasmic tunnel. These proteins generate heterogeneous pores that facilitate the release of vRNAP. We further uncover a signaling hub in the phage tail, composed of the receptor-binding protein, tail tube, and tail plug, that detects receptor engagement and orchestrates the release of ejection proteins. We propose that the beads-on-a-string architecture of vRNAP enables the translocation of megadalton-scale protein complexes through the ~35 Å channel formed by the tail and ejection proteins. These findings establish N4 as a distinctive model for protein translocation through biological channels.

Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complimentary determining region 2 (CDR2) of a soluble, clinically validated nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered CALR-Nb02 by adapting the core of Cal3 to a partial consensus framework sequence of stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a predominantly monomeric, soluble protein that exhibits an increased thermal stability and a higher calreticulin binding affinity compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody, and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.

Mycobacterium tuberculosis uses several ESX type VII protein secretion systems for pathogenesis. M. tuberculosis ESX-5 is only partially characterized because it is essential for growth in standard lab culture conditions. To circumvent ESX-5 essentiality, we made an M. tuberculosis strain in which the central ESX-5 membrane component EccD ₅ can be conditionally depleted. Here, we use this strain to demonstrate that M. tuberculosis requires the ESX-5 secretion system to grow using specific carbon sources in vitro , to grow in cultured macrophages, and to replicate and disseminate in aerosol-infected mice. M. tuberculosis requires ESX-5 to use glycerol or glucose as the sole carbon source. Use of glycerol and glucose also depends on the outer membrane protein PPE51. We show that M. tuberculosis requires ESX-5 activity for outer membrane export and surface exposure of PPE51. Expression of the outer membrane porin MspA enabled growth of ESX-5-deficient M. tuberculosis on glycerol, suggesting that the main function of ESX-5 in vitro is to export nutrient transporters to the outer membrane. Depletion of EccD ₅ in acutely infected mice caused clearance of M. tuberculosis from lung tissues, demonstrating the critical importance of ESX-5 activity during infection. Our findings suggest that ESX-5 promotes M. tuberculosis pathogenesis by mediating export of outer membrane proteins that enable nutrient acquisition. IMPORTANCE: Mycobacterium tuberculosis ESX type VII secretion systems play important roles in pathogenesis, but the functions of ESX-5 are not well characterized because it is essential for growth in standard lab culture conditions. We used a strain that conditionally expresses a central membrane component of the ESX-5 secretion apparatus to determine how ESX-5 impacts growth in lab cultures and in a mouse infection model. We found that M. tuberculosis requires ESX-5 to grow using several carbon sources and to grow in the lungs of infected mice. Inhibiting production of the ESX-5 secretion system in mice also led to clearance of M. tuberculosis from lung tissues. Our results demonstrate that the M. tuberculosis ESX-5 system is a critical virulence factor and suggest that ESX-5 is a strong candidate for antitubercular drug development.

Mycobacterium tuberculosis contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners MmpS4 and MmpS5, export the mycobacterial siderophore mycobactin and the last resort TB drug bedaquiline. Recently, we determined a structure of the MmpL4 monomer in complex with desferrated mycobactin, which lacked a functionally essential coiled-coil domain predicted to extend far into the periplasm. Here, we present a cryo-EM structure of the hexameric (MmpS4)3-(MmpL4)3 complex, which was enabled by rational disulfide cross-links based on AlphaFold predictions. We observed density for the coiled-coil domain, which protrudes into the periplasmic space at an angle of around 60° relative to the symmetry axis of the MmpL4 trimer. In the context of the hexameric complex, MmpL4’s conformation differs strikingly from the one observed for monomeric MmpL4, which includes formation of a large cavity in the periplasmic domain and rearrangements of conserved proton coupling residues at the transmembrane domain. Our work provides an experimental workflow to obtain single particle cryo-EM structures of labile multiprotein complexes by AlphaFold-informed stabilization of predicted protein interfaces.

Calreticulin is an emerging cancer biomarker, but current detection methods rely on expensive monoclonal antibodies that suffer from inefficient protein production, pharmacokinetic challenges and poor tissue penetration. Cal3, a calreticulin-specific nanobody, was constructed by replacing the complementarity-determining region 2 (CDR2) of a soluble, clinically validated HER2 nanobody with a calreticulin-specific CDR2 isolated from a phage display library. However, the poor solubility and low yield of Cal3 limit its usefulness. In this study, we engineered the CALR nanobody 02 by adapting the core of Cal3 to a consensus framework sequence for stable nanobodies. CALR-Nb02 was purified with a 240-fold higher yield as a soluble protein with an increased thermal stability and a five-fold higher calreticulin binding affinity (KD: 25 nM) compared with Cal3. These results reveal a strategy for quickly altering the specificity of a stable nanobody and provide an improved calreticulin-binding reagent for future diagnostic, imaging, and therapeutic applications.

Mycobacterium tuberculosis contains thirteen Mycobacterial membrane protein Large (MmpL) transporters, which belong to the family of secondary active RND transporters. MmpL4 and MmpL5, together with their operon partners MmpS4 and MmpS5, export the mycobacterial siderophore mycobactin and the last resort TB drug bedaquiline. Recently, we determined a structure of the MmpL4 monomer in complex with desferrated mycobactin, which lacked a functionally essential coiled-coil domain predicted to extend far into the periplasm. Here, we present a cryo-EM structure of the hexameric (MmpS4)3-(MmpL4)3 complex, which was enabled by rational disulfide cross-links based on AlphaFold predictions. We observed density for the coiled-coil domain, which protrudes into the periplasmic space at an angle of around 60° relative to the symmetry axis of the MmpL4 trimer. In the context of the hexameric complex, MmpL4’s conformation differs strikingly from the one observed for monomeric MmpL4, which includes formation of a large cavity in the periplasmic domain and rearrangements of conserved proton coupling residues at the transmembrane domain. Our work provides an experimental workflow to obtain single particle cryo-EM structures of labile multiprotein complexes by AlphaFold-informed stabilization of predicted protein interfaces.

Mycobacterium tuberculosis uses five ESX systems to secrete effector proteins with distinct functions essential for the pathogen's growth and virulence. The current paradigm assumes that each ESX system secretes a distinct set of effector proteins. Here, we show that the EsxU-EsxT proteins, associated with the ESX-4 system, and the EsxE-EsxF proteins, encoded in the toxin operon cpnT, form a supercomplex that is required not only for toxin secretion, but also for outer membrane localization and for secretion of all Esx proteins into the cytosol of infected macrophages. Secretion of non-Esx proteins such as EspB, proteins involved in iron uptake, and PPE62, which are dependent on ESX-1, ESX-3 and ESX-5, respectively, also requires the EsxUT-EsxEF supercomplex. The mutual functional dependency of EsxEF and EsxUT on each other for outer-membrane localization synchronizes effector protein secretion, enabling M. tuberculosis to block phagosomal maturation and to permeabilize the phagosomal membrane when the pathogen is capable of killing host cells by toxin secretion. The requirement of the ESX-4 system for general secretion of ESX-dependent proteins in vivo is a critical vulnerability that could be targeted by drugs and/or in vaccines to simultaneously block many virulence factors of M. tuberculosis.

Mycobacterium tuberculosis uses several ESX type VII protein secretion systems for pathogenesis. M. tuberculosis ESX-5 is poorly characterized because it is essential for growth in standard lab culture conditions. To circumvent ESX-5 essentiality, we made an M. tuberculosis strain in which the central ESX-5 membrane component EccD ₅ can be conditionally depleted. Here, we use this strain to demonstrate that M. tuberculosis requires the ESX-5 secretion system to grow using specific carbon sources in vitro , to grow in cultured macrophages, and to replicate and disseminate in aerosol-infected mice. M. tuberculosis requires ESX-5 to use glycerol or glucose as the sole carbon source. Use of glycerol and glucose also depends on the outer membrane protein PPE51. We show that M. tuberculosis requires ESX-5 activity for outer membrane export and surface exposure of PPE51. Expression of the outer membrane porin MspA enabled growth of ESX-5 deficient M. tuberculosis on glycerol, suggesting that the main function of ESX-5 in vitro is to export nutrient transporters to the outer membrane. Importantly, depletion of EccD ₅ in acutely infected mice resulted in clearance of M. tuberculosis from lung tissues, demonstrating the critical importance of ESX-5 activity during infection. Our findings suggest that ESX-5 promotes M. tuberculosis pathogenesis by mediating export of outer membrane proteins that enable nutrient acquisition. Importance: Mycobacterium tuberculosis ESX type VII secretion systems play important roles in pathogenesis, but the functions of ESX-5 are not well characterized because it is essential for growth in standard lab culture conditions. We used a strain that conditionally expresses a central membrane component of the ESX-5 secretion apparatus to determine how ESX-5 impacts growth in lab cultures and in a mouse infection model. We found that M. tuberculosis requires ESX-5 to grow using several carbon sources and to grow in the lungs of infected mice. Inhibiting production of the ESX-5 secretion system in mice also led to clearance of M. tuberculosis from lung tissues. Our results demonstrate that the M. tuberculosis ESX-5 system is a critical virulence factor and suggest that ESX-5 is a strong candidate for anti-tubercular drug development.