Axon Degeneration Gated by Retrograde Activation of Somatic Pro-apoptotic Signaling.

During development, sensory axons compete for limiting neurotrophic support, and local neurotrophin insufficiency triggers caspase-dependent axon degeneration. The signaling driving axon degeneration upon local deprivation is proposed to reside within axons. Our results instead support a model in which, despite the apoptotic machinery being present in axons, the cell body is an active participant in gating axonal caspase activation and axon degeneration. Loss of trophic support in axons initiates retrograde activation of a somatic pro-apoptotic pathway, which, in turn, is required for distal axon degeneration via an anterograde pro-degenerative factor. At a molecular level, the cell body is the convergence point of two signaling pathways whose integrated action drives upregulation of pro-apoptotic Puma, which, unexpectedly, is confined to the cell body. Puma then overcomes inhibition by pro-survival Bcl-xL and Bcl-w and initiates the anterograde pro-degenerative program, highlighting the role of the cell body as an arbiter of large-scale axon removal.

Germline NLRP1 Mutations Cause Skin Inflammatory and Cancer Susceptibility Syndromes via Inflammasome Activation.

Inflammasome complexes function as key innate immune effectors that trigger inflammation in response to pathogen- and danger-associated signals. Here, we report that germline mutations in the inflammasome sensor NLRP1 cause two overlapping skin disorders: multiple self-healing palmoplantar carcinoma (MSPC) and familial keratosis lichenoides chronica (FKLC). We find that NLRP1 is the most prominent inflammasome sensor in human skin, and all pathogenic NLRP1 mutations are gain-of-function alleles that predispose to inflammasome activation. Mechanistically, NLRP1 mutations lead to increased self-oligomerization by disrupting the PYD and LRR domains, which are essential in maintaining NLRP1 as an inactive monomer. Primary keratinocytes from patients experience spontaneous inflammasome activation and paracrine IL-1 signaling, which is sufficient to cause skin inflammation and epidermal hyperplasia. Our findings establish a group of non-fever inflammasome disorders, uncover an unexpected auto-inhibitory function for the pyrin domain, and provide the first genetic evidence linking NLRP1 to skin inflammatory syndromes and skin cancer predisposition.

MAJIN Links Telomeric DNA to the Nuclear Membrane by Exchanging Telomere Cap.

In meiosis, telomeres attach to the inner nuclear membrane (INM) and drive the chromosome movement required for homolog pairing and recombination. Here, we address the question of how telomeres are structurally adapted for the meiotic task. We identify a multi-subunit meiotic telomere-complex, TERB1/2-MAJIN, which takes over telomeric DNA from the shelterin complex in mouse germ cells. TERB1/2-MAJIN initially assembles on the INM sequestered by its putative transmembrane subunit MAJIN. In early meiosis, telomere attachment is achieved by the formation of a chimeric complex of TERB1/2-MAJIN and shelterin. The chimeric complex matures during prophase into DNA-bound TERB1/2-MAJIN by releasing shelterin, forming a direct link between telomeric DNA and the INM. These hierarchical processes, termed "telomere cap exchange," are regulated by CDK-dependent phosphorylation and the DNA-binding activity of MAJIN. Further, we uncover a positive feedback between telomere attachment and chromosome movement, revealing a comprehensive regulatory network underlying meiosis-specific telomere function in mammals.

Immunoglobulin M perception by FcµR.

Immunoglobulin M (IgM) is the first antibody to emerge during embryonic development and the humoral immune response1. IgM can exist in several distinct forms, including monomeric, membrane-bound IgM within the B cell receptor (BCR) complex, pentameric and hexameric IgM in serum and secretory IgM on the mucosal surface. FcµR, the only IgM-specific receptor in mammals, recognizes different forms of IgM to regulate diverse immune responses2-5. However, the underlying molecular mechanisms remain unknown. Here we delineate the structural basis of the FcµR-IgM interaction by crystallography and cryo-electron microscopy. We show that two FcµR molecules interact with a Fcµ-Cµ4 dimer, suggesting that FcµR can bind to membrane-bound IgM with a 2:1 stoichiometry. Further analyses reveal that FcµR-binding sites are accessible in the context of IgM BCR. By contrast, pentameric IgM can recruit four FcµR molecules to bind on the same side and thereby facilitate the formation of an FcµR oligomer. One of these FcµR molecules occupies the binding site of the secretory component. Nevertheless, four FcµR molecules bind to the other side of secretory component-containing secretory IgM, consistent with the function of FcµR in the retrotransport of secretory IgM. These results reveal intricate mechanisms of IgM perception by FcµR.

Crystal Structures of the Full-Length Murine and Human Gasdermin D Reveal Mechanisms of Autoinhibition, Lipid Binding, and Oligomerization.

Gasdermin D (GSDMD) is an effector molecule for pyroptosis downstream of canonical and noncanonical inflammasome signaling pathways. Cleavage of GSDMD by inflammatory caspases triggers the oligomerization and lipid binding by its N-terminal domain, which assembles membrane pores, whereas its C-terminal domain binds the N-terminal domain to inhibit pyroptosis. Despite recent progress in our understanding of the structure and function of the murine gasdermin A3 (mGSDMA3), the molecular mechanisms of GSDMD activation and regulation remain poorly characterized. Here, we report the crystal structures of the full-length murine and human GSDMDs, which reveal the architecture of the GSDMD N-terminal domains and demonstrate distinct and common features of autoinhibition among gasdermin family members utilizing their ß1-ß2 loops. Disruption of the intramolecular domain interface enhanced pyroptosis, whereas mutations at the predicted lipid-binding or oligomerization surface reduced cytolysis. Our study provides a framework for understanding the autoinhibition, lipid binding, and oligomerization of GSDMD by using overlapping interfaces.

Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.

Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. Here, we show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the nonautophagy Vps34 complex by phosphorylating T163/S165 in Vps34 and therefore suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the proautophagy Vps34 complex by phosphorylating S91/S94 in Beclin1 to induce autophagy. Atg14L, an autophagy-essential gene present only in the proautophagy Vps34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK in nutrient stress response and autophagy.

Beclin 2 functions in autophagy, degradation of G protein-coupled receptors, and metabolism.

The molecular mechanism of autophagy and its relationship to other lysosomal degradation pathways remain incompletely understood. Here, we identified a previously uncharacterized mammalian-specific protein, Beclin 2, which, like Beclin 1, functions in autophagy and interacts with class III PI3K complex components and Bcl-2. However, Beclin 2, but not Beclin 1, functions in an additional lysosomal degradation pathway. Beclin 2 is required for ligand-induced endolysosomal degradation of several G protein-coupled receptors (GPCRs) through its interaction with GASP1. Beclin 2 homozygous knockout mice have decreased embryonic viability, and heterozygous knockout mice have defective autophagy, increased levels of brain cannabinoid 1 receptor, elevated food intake, and obesity and insulin resistance. Our findings identify Beclin 2 as a converging regulator of autophagy and GPCR turnover and highlight the functional and mechanistic diversity of Beclin family members in autophagy, endolysosomal trafficking, and metabolism.

Nasal delivery of an IgM offers broad protection from SARS-CoV-2 variants.

Resistance represents a major challenge for antibody-based therapy for COVID-191-4. Here we engineered an immunoglobulin M (IgM) neutralizing antibody (IgM-14) to overcome the resistance encountered by immunoglobulin G (IgG)-based therapeutics. IgM-14 is over 230-fold more potent than its parental IgG-14 in neutralizing SARS-CoV-2. IgM-14 potently neutralizes the resistant virus raised by its corresponding IgG-14, three variants of concern-B.1.1.7 (Alpha, which first emerged in the UK), P.1 (Gamma, which first emerged in Brazil) and B.1.351 (Beta, which first emerged in South Africa)-and 21 other receptor-binding domain mutants, many of which are resistant to the IgG antibodies that have been authorized for emergency use. Although engineering IgG into IgM enhances antibody potency in general, selection of an optimal epitope is critical for identifying the most effective IgM that can overcome resistance. In mice, a single intranasal dose of IgM-14 at 0.044 mg per kg body weight confers prophylactic efficacy and a single dose at 0.4 mg per kg confers therapeutic efficacy against SARS-CoV-2. IgM-14, but not IgG-14, also confers potent therapeutic protection against the P.1 and B.1.351 variants. IgM-14 exhibits desirable pharmacokinetics and safety profiles when administered intranasally in rodents. Our results show that intranasal administration of an engineered IgM can improve efficacy, reduce resistance and simplify the prophylactic and therapeutic treatment of COVID-19.

Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy.

The BCL-2 protein family determines the commitment of cells to apoptosis, an ancient cell suicide programme that is essential for development, tissue homeostasis and immunity. Too little apoptosis can promote cancer and autoimmune diseases; too much apoptosis can augment ischaemic conditions and drive neurodegeneration. We discuss the biochemical, structural and genetic studies that have clarified how the interplay between members of the BCL-2 family on mitochondria sets the apoptotic threshold. These mechanistic insights into the functions of the BCL-2 family are illuminating the physiological control of apoptosis, the pathological consequences of its dysregulation and the promising search for novel cancer therapies that target the BCL-2 family.

The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase.

XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.

Reconciling ASPP-p53 binding mode discrepancies through an ensemble binding framework that bridges crystallography and NMR data.

ASPP2 and iASPP bind to p53 through their conserved ANK-SH3 domains to respectively promote and inhibit p53-dependent cell apoptosis. While crystallography has indicated that these two proteins employ distinct surfaces of their ANK-SH3 domains to bind to p53, solution NMR data has suggested similar surfaces. In this study, we employed multi-scale molecular dynamics (MD) simulations combined with free energy calculations to reconcile the discrepancy in the binding modes. We demonstrated that the binding mode based solely on a single crystal structure does not enable iASPP's RT loop to engage with p53's C-terminal linker-a verified interaction. Instead, an ensemble of simulated iASPP-p53 complexes facilitates this interaction. We showed that the ensemble-average inter-protein contacting residues and NMR-detected interfacial residues qualitatively overlap on ASPP proteins, and the ensemble-average binding free energies better match experimental KD values compared to single crystallgarphy-determined binding mode. For iASPP, the sampled ensemble complexes can be grouped into two classes, resembling the binding modes determined by crystallography and solution NMR. We thus propose that crystal packing shifts the equilibrium of binding modes towards the crystallography-determined one. Lastly, we showed that the ensemble binding complexes are sensitive to p53's intrinsically disordered regions (IDRs), attesting to experimental observations that these IDRs contribute to biological functions. Our results provide a dynamic and ensemble perspective for scrutinizing these important cancer-related protein-protein interactions (PPIs).

Structural transitions in TCTP tumor protein upon binding to the anti-apoptotic protein family member Mcl-1.

Translationally Controlled Tumor Protein (TCTP) serves as a pro-survival factor in tumor cells, inhibiting the mitochondrial apoptosis pathway by enhancing the function of anti-apoptotic Bcl-2 family members Mcl-1 and Bcl-xL. TCTP specifically binds to Bcl-xL, preventing Bax-dependent Bcl-xL-induced cytochrome c release, and it reduces Mcl-1 turnover by inhibiting its ubiquitination, thereby decreasing Mcl-1-mediated apoptosis. TCTP harbors a BH3-like motif that forms a ß-strand buried in the globular domain of the protein. In contrast, the crystal structure of the TCTP BH3-like peptide in complex with the Bcl-2 family member Bcl-xL reveals an α-helical conformation for the BH3-like motif, suggesting significant structural changes upon complex formation. Employing biochemical and biophysical methods, including limited proteolysis, circular dichroism, NMR, and SAXS, we describe the TCTP complex with the Bcl-2 homolog Mcl-1. Our findings demonstrate that full-length TCTP binds to the BH3 binding groove of Mcl-1 via its BH3-like motif, experiencing conformational exchange at the interface on a micro- to milli-second timescale. Concurrently, the TCTP globular domain becomes destabilized, transitioning into a molten-globule state. Furthermore, we establish that the non-canonical residue D16 within the TCTP BH3-like motif reduces stability while enhancing the dynamics of the intermolecular interface. In conclusion, we detail the structural plasticity of TCTP and discuss its implications for partner interactions and future anticancer drug design strategies aimed at targeting TCTP complexes.

Identification of nonhistone substrates of the lysine methyltransferase PRDM9.

Lysine methylation is a dynamic, posttranslational mark that regulates the function of histone and nonhistone proteins. Many of the enzymes that mediate lysine methylation, known as lysine methyltransferases (KMTs), were originally identified to modify histone proteins but have also been discovered to methylate nonhistone proteins. In this work, we investigate the substrate selectivity of the KMT PRDM9 to identify both potential histone and nonhistone substrates. Though normally expressed in germ cells, PRDM9 is significantly upregulated across many cancer types. The methyltransferase activity of PRDM9 is essential for double-strand break formation during meiotic recombination. PRDM9 has been reported to methylate histone H3 at lysine residues 4 and 36; however, PRDM9 KMT activity had not previously been evaluated on nonhistone proteins. Using lysine-oriented peptide libraries to screen potential substrates of PRDM9, we determined that PRDM9 preferentially methylates peptide sequences not found in any histone protein. We confirmed PRDM9 selectivity through in vitro KMT reactions using peptides with substitutions at critical positions. A multisite λ-dynamics computational analysis provided a structural rationale for the observed PRDM9 selectivity. The substrate selectivity profile was then used to identify putative nonhistone substrates, which were tested by peptide spot array, and a subset was further validated at the protein level by in vitro KMT assays on recombinant proteins. Finally, one of the nonhistone substrates, CTNNBL1, was found to be methylated by PRDM9 in cells.

PEG10 viral aspartic protease domain is essential for the maintenance of fetal capillary structure in the mouse placenta.

The therian-specific gene paternally expressed 10 (Peg10) plays an essential role in placenta formation: Peg10 knockout mice exhibit early embryonic lethality as a result of severe placental defects. The PEG10 protein exhibits homology with long terminal repeat (LTR) retrotransposon GAG and POL proteins; therefore, we generated mice harboring a mutation in the highly conserved viral aspartic protease motif in the POL-like region of PEG10 because this motif is essential for the life cycle of LTR retrotransposons/retroviruses. Intriguingly, frequent perinatal lethality, not early embryonic lethality, was observed with fetal and placental growth retardation starting mid-gestation. In the mutant placentas, severe defects were observed in the fetal vasculature, where PEG10 is expressed in the three trophoblast cell layers that surround fetal capillary endothelial cells. Thus, Peg10 has essential roles, not only in early placenta formation, but also in placental vasculature maintenance from mid- to late-gestation. This implies that along the feto-maternal placenta interface an interaction occurs between two retrovirus-derived genes, Peg10 and retrotransposon Gag like 1 (Rtl1, also called Peg11), that is essential for the maintenance of fetal capillary endothelial cells.

The N-end rule ubiquitin ligase UBR2 mediates NLRP1B inflammasome activation by anthrax lethal toxin.

Anthrax lethal toxin (LT) is known to induce NLRP1B inflammasome activation and pyroptotic cell death in macrophages from certain mouse strains in its metalloprotease activity-dependent manner, but the underlying mechanism is unknown. Here, we establish a simple but robust cell system bearing dual-fluorescence reporters for LT-induced ASC specks formation and pyroptotic lysis. A genome-wide siRNA screen and a CRISPR-Cas9 knockout screen were applied to this system for identifying genes involved in LT-induced inflammasome activation. UBR2, an E3 ubiquitin ligase of the N-end rule degradation pathway, was found to be required for LT-induced NLRP1B inflammasome activation. LT is known to cleave NLRP1B after Lys44. The cleaved NLRP1B, bearing an N-terminal leucine, was targeted by UBR2-mediated ubiquitination and degradation. UBR2 partnered with an E2 ubiquitin-conjugating enzyme UBE2O in this process. NLRP1B underwent constitutive autocleavage before the C-terminal CARD domain. UBR2-mediated degradation of LT-cleaved NLRP1B thus triggered release of the noncovalent-bound CARD domain for subsequent caspase-1 activation. Our study illustrates a unique mode of inflammasome activation in cytosolic defense against bacterial insults.

A Step Forward toward Selective Activation/Inhibition of Bak, a Pro-Apoptotic Member of the Bcl-2 Protein Family: Discovery of New Prospective Allosteric Sites Using Molecular Dynamics.

Bak is a pro-apoptotic protein and a member of the Bcl-2 family that plays a key role in apoptosis, a programmed cell death mechanism of multicellular organisms. Its activation under death stimuli triggers the permeabilization of the mitochondrial outer membrane that represents a point of no return in the apoptotic pathway. This process is deregulated in many tumors where Bak is inactivated, whereas in other cases like in neurodegeneration, it exhibits an excessive response leading to disorders such as the Alzheimer disease. Members of the Bcl-2 family share a common 3D structure, exhibiting an extremely similar orthosteric binding site, a place where both pro and antiapoptotic proteins bind. This similarity raises a selectivity issue that hampers the identification of new drugs, capable of altering Bak activation in a selective manner. An alternative activation site triggered by antibodies has been recently identified, opening the opportunity to undertake new drug discovery studies. Despite this recent identification, an exhaustive study to identify cryptic pockets as prospective allosteric sites has not been yet performed. Thus, the present study aims to characterize novel hotspots in the Bak structure. For this purpose, we have carried out extensive molecular dynamics simulations using three different Bak systems including Bak in its apo form, Bak in complex with its endogen activator Bim and an intermediate form, set up by removing Bim from the previous complex. The results reported in the present work shed some light on future docking studies on Bak through the identification of new prospective allosteric sites, not previously described in this protein.

Engineered variants of the Ras effector protein RASSF5 (NORE1A) promote anticancer activities in lung adenocarcinoma.

Within the superfamily of small GTPases, Ras appears to be the master regulator of such processes as cell cycle progression, cell division, and apoptosis. Several oncogenic Ras mutations at amino acid positions 12, 13, and 61 have been identified that lose their ability to hydrolyze GTP, giving rise to constitutive signaling and eventually development of cancer. While disruption of the Ras/effector interface is an attractive strategy for drug design to prevent this constitutive activity, inhibition of this interaction using small molecules is impractical due to the absence of a cavity to which such molecules could bind. However, proteins and especially natural Ras effectors that bind to the Ras/effector interface with high affinity could disrupt Ras/effector interactions and abolish procancer pathways initiated by Ras oncogene. Using a combination of computational design and in vitro evolution, we engineered high-affinity Ras-binding proteins starting from a natural Ras effector, RASSF5 (NORE1A), which is encoded by a tumor suppressor gene. Unlike previously reported Ras oncogene inhibitors, the proteins we designed not only inhibit Ras-regulated procancer pathways, but also stimulate anticancer pathways initiated by RASSF5. We show that upon introduction into A549 lung carcinoma cells, the engineered RASSF5 mutants decreased cell viability and mobility to a significantly greater extent than WT RASSF5. In addition, these mutant proteins induce cellular senescence by increasing acetylation and decreasing phosphorylation of p53. In conclusion, engineered RASSF5 variants provide an attractive therapeutic strategy able to oppose cancer development by means of inhibiting of procancer pathways and stimulating anticancer processes.

Dynamic multimerization of Dab2-Myosin VI complexes regulates cargo processivity while minimizing cortical actin reorganization.

Myosin VI ensembles on endocytic cargo facilitate directed transport through a dense cortical actin network. Myosin VI is recruited to clathrin-coated endosomes via the cargo adaptor Dab2. Canonically, it has been assumed that the interactions between a motor and its cargo adaptor are stable. However, it has been demonstrated that the force generated by multiple stably attached motors disrupts local cytoskeletal architecture, potentially compromising transport. In this study, we demonstrate that dynamic multimerization of myosin VI-Dab2 complexes facilitates cargo processivity without significant reorganization of cortical actin networks. Specifically, we find that Dab2 myosin interacting region (MIR) binds myosin VI with a moderate affinity (184 nM) and single-molecule kinetic measurements demonstrate a high rate of turnover (1 s-1) of the Dab2 MIR-myosin VI interaction. Single-molecule motility shows that saturating Dab2-MIR concentration (2 µM) promotes myosin VI homodimerization and processivity with run lengths comparable with constitutive myosin VI dimers. Cargo-mimetic DNA origami scaffolds patterned with Dab2 MIR-myosin VI complexes are weakly processive, displaying sparse motility on single actin filaments and "stop-and-go" motion on a cellular actin network. On a minimal actin cortex assembled on lipid bilayers, unregulated processive movement by either constitutive myosin V or VI dimers results in actin remodeling and foci formation. In contrast, Dab2 MIR-myosin VI interactions preserve the integrity of a minimal cortical actin network. Taken together, our study demonstrates the importance of dynamic motor-cargo association in enabling cargo transportation without disrupting cytoskeletal organization.

Structural evidence that MOAP1 and PEG10 are derived from retrovirus/retrotransposon Gag proteins.

The Gag proteins of retroviruses play an essential role in virus particle assembly by forming a protein shell or capsid and thus generating the virion compartment. A variety of human proteins have now been identified with structural similarity to one or more of the major Gag domains. These human proteins are thought to have been evolved or "domesticated" from ancient integrations due to retroviral infections or retrotransposons. Here, we report that X-ray crystal structures of stably folded domains of MOAP1 (modulator of apoptosis 1) and PEG10 (paternally expressed gene 10) are highly similar to the C-terminal capsid (CA) domains of cognate Gag proteins. The structures confirm classification of MOAP1 and PEG10 as domesticated Gags, and suggest that these proteins may have preserved some of the key interactions that facilitated assembly of their ancestral Gags into capsids.

In silico structure evaluation of BAG3 and elucidating its association with bacterial infections through protein-protein and host-pathogen interaction analysis.

BAG3, a co-chaperone protein with a Bcl-2-associated athanogene (BAG) domain, has diverse functionalities in protein-folding, apoptosis, inflammation, and cell cycle regulatory cross-talks. It has been well characterised in cardiac diseases, cancers, and viral pathogenesis. The multiple roles of BAG3 are attributed to its functional regions like BAG, Tryptophan-rich (WW), isoleucine-proline-valine-rich (IPV), and proline-rich (PXXP) domains. However, to study its structural impact on various functions, the experimental 3D structure of BAG3 protein was not available. Hence, the structure was predicted through in silico modelling and validated through computational tools and molecular dynamics simulation studies. To the best of our knowledge, the role of BAG3 in bacterial infections is not explicitly reported. We attempted to study them through an in-silico protein-protein interaction network and host-pathogen interaction analysis. From structure-function relationships, it was identified that the WW and PXXP domains were associated with cellular cytoskeleton rearrangement and adhesion-mediated response, which might be involved in BAG3-related intracellular bacterial proliferation. From functional enrichment analysis, Gene Ontology terms and topological matrices, 18 host proteins and 29 pathogen proteins were identified in the BAG3 interactome pertaining to Legionellosis, Tuberculosis, Salmonellosis, Shigellosis, and Pertussis through differential phosphorylation events associated with serine metabolism. Furthermore, it was evident that direct (MAPK8, MAPK14) and associated (MAPK1, HSPD1, NFKBIA, TLR2, RHOA) interactors of BAG3 could be considered as therapeutic markers to curb down intracellular bacterial propagation in humans.