Infected Host Cells Research Articles

Early Use of Liraglutide for the Treatment of Acute COVID-19 Infection: An Open-Label Single-Center Phase II Safety Study with Biomarker Profiling

Background: Glucagon-like peptide-1 (GLP-1) agonists are an existing treatment option for patients with insulin-resistant states, which elicit further pleiotropic effects related to immune cell recruitment and vascular inflammation. GLP-1 agonists downregulate the cluster of differentiation 147 (CD147) receptor, one of several receptors for the SARS-CoV-2 spike protein that mediate viral infection of host cells. Methods: We conducted an open-label prospective safety and tolerability study including biomarker responses of the GLP-1 agonist Liraglutide, administered for 5 days as an add-on therapy to the standard of care within 48 h of presentation in a cohort of 13 patients hospitalized with COVID-19 pneumonia. Biomarker responses were compared in patients admitted to critical care and those not requiring critical care admission (non-critical group). Results: Liraglutide (0.6 mg, subcutaneously) was well tolerated by all patients and all patients were alive 30 days after diagnosis. Plasma soluble CD147 levels were reduced in the non-critical patient group at day 5 in contrast to critical care-treated patients, who demonstrated an increase in soluble CD147 levels between day 0 and day 5. Patients with milder COVID-19 pneumonia severity also demonstrated improvement in echocardiographic parameters of right and left ventricular function, reduction in plasma Troponin levels, increased CD147 expression on T lymphocytes, and reduction in plasma IL-8. Conclusions: This first-in-disease use of the GLP-1 agonist Liraglutide demonstrates its safety and tolerability in an unselected cohort of patients hospitalized with COVID-19 pneumonia across a range of clinical severities.

Identification and subcellular localization of proteins that interact with Duck plague virus pUL14 in infected host cells.

Duck plague (DP), which is caused by duck plague virus (DPV), is an infectious disease that severely harms the waterfowl breeding industry. The UL14 protein (pUL14) is a tegument protein encoded by the UL14 gene, which is located in the unique long (UL) region of the DPV genome. DPV pUL14 plays a crucial role in viral replication, likely by interacting with host and viral proteins that have yet to be identified. In this study, glutathione-S-transferase (GST) pull-down combined with liquid chromatography-mass spectrometry (LC-MS/MS) was employed to identify pUL14-interacting proteins in DPV-infected cells. A total of 281 host proteins and 58 viral proteins that interacted with pUL14 were identified. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the identified proteins could be assigned to several different subcellular locations and functional classes. These proteins are associated mainly with the regulation of biological processes, RNA biosynthetic processes, and nuclear export. In addition, four viral proteins of interest, the α-gene transducing factor (α-TIF) pUL48, the nuclear egress complex (NEC) proteins pUL31/34, and pUL51, a protein involved in secondary envelopment, were validated by coimmunoprecipitation (co-IP) to interact with DPV pUL14. Additionally, the nuclear export signal (NES) was identified in a leucine-rich region at aa 77-87 (77VQTKIEEQLAI87) of DPV pUL14. The interactome data between DPV pUL14 and host/viral proteins contribute to understanding the role of DPV pUL14 in the replication of DPV.

Engineered Phage Enables Efficient Control of Gene Expression upon Infection of the Host Cell.

Recently, we developed a spatial phage-assisted continuous evolution (SPACE) system. This system utilizes chemotaxis coupled with the growth of motile bacteria during their spatial range expansion in soft agar to provide fresh host cells for iterative phage infection and selection pressure for preserving evolved genes of interest carried by phage mutants. Controllable mutagenesis activated only in a subpopulation of the migrating cells is essential in this system to efficiently generate mutated progeny phages from which desired individuals are selected during the directed evolution process. But, the widely adopted small molecule-dependent inducible system could hardly fulfill this purpose because it always affects all cells homogeneously. In this study, we developed a phage infection-induced gene expression system using modified Escherichia coli (E. coli) phage shock protein operon or sigma factors from Bacillus subtilis. Results showed that this system enabled efficient control of gene expression upon phage infection with dynamic output ranges from small to large using combinations of different engineered phages and corresponding promoters. This system was incorporated into SPACE to function as a phage infection-induced mutagenesis module and successfully facilitated the evolution of T7 RNA polymerase, which generated diverse mutants with altered promoter recognition specificity. We expect that phage infection-induced gene expression system could be further extended to more applications involving partial induction in a portion of a population and targeted induction in specific strains among a mixed bacterial community, which provides an important complement to small molecule-dependent inducible systems.

Glutamine metabolism Is essential for coronavirus replication in host cells and in mice.

Understanding the fundamental biochemical and metabolic requirements for the replication of coronaviruses within infected cells is of notable interest for the development of broad-based therapeutic strategies, given the likelihood of emergence of new pandemic-potential virus species, as well as future variants of SARS-CoV-2. Here we demonstrate members of the glutaminase family of enzymes (GLS and GLS2), which catalyze the hydrolysis of glutamine to glutamate (i.e., the first step in glutamine metabolism), play key roles in coronavirus replication in host cells. Using a range of human seasonal and zoonotic coronaviruses, we show three examples where GLS expression increases during coronavirus infection of host cells, and another where GLS2 is upregulated. The viruses hijack the metabolic machinery responsible for glutamine metabolism to generate the building blocks for biosynthetic processes and satisfy the bioenergetic requirements demanded by the 'glutamine addiction' of virus-infected cells. We demonstrate that genetic silencing of glutaminase enzymes reduces coronavirus infection and that newer members of two classes of allosteric inhibitors targeting these enzymes, designated as SU1, a pan-GLS/GLS2 inhibitor, and UP4, a specific GLS inhibitor, block viral replication in epithelial cells. Moreover, treatment of SARS-CoV-2 infected K18-human ACE2 transgenic mice with SU1 resulted in their complete survival compared to untreated control animals, which succumbed within 10 days post-infection. Overall, these findings highlight the importance of glutamine metabolism for coronavirus replication in human cells and mice and show that glutaminase inhibitors can block coronavirus infection and thereby may represent a novel class of broad-based anti-viral drug candidates.

Brussonol and komaroviquinone as inhibitors of the SARS-CoV-2 Omicron BA.2 variant spike protein: A molecular docking, molecular dynamics, and quantum biochemistry approach

Since late 2019, humanity has faced the challenges posed by the COVID-19 pandemic, caused by the SARS-CoV-2 virus. The continuous evolution of SARS-CoV-2 has led to the emergence of multiple Variants of Concern (VOCs) and Variants of Interest (VOIs), posing significant risks to global health. SARS-CoV-2 infects host cells via the angiotensin-converting enzyme 2 (ACE2) receptors, facilitated by the spike (S) protein. Icetexane diterpenes, including brussonol and komaroviquinone, exhibit notable anti-inflammatory, antibacterial, antiviral, antiproliferative, and anticancer properties. Recent research has explored their potential as inhibitors of the SARS-CoV-2 3Clpro protease, showing promising efficacy comparable to Nirmatrelvir. This study investigates brussonol and komaroviquinone as potential inhibitors of the SARS-CoV-2 Omicron BA.2 variant spike protein using molecular docking, molecular dynamics simulations, and quantum biochemistry approaches. The stability and interaction energies of brussonol, komaroviquinone, and mefloquine with the SARS-CoV-2 Omicron BA.2 variant spike protein were evaluated. RMSD analysis demonstrated that komaroviquinone and mefloquine maintain more stable binding poses with the spike protein compared to various NAGs and glycans. Electrostatic potential maps revealed significant interactions with ASN603, a critical residue for ligand binding efficacy. Furthermore, this study addresses a gap in current research, as no studies were found that simulate the trimer of the SARS-CoV-2 BA.2 variant spike protein. Most existing studies focus on the monomer and often exclude the NAGs and glycans. This research underscores the importance of maintaining the NAGs and glycans in the trimer simulations, providing a more accurate representation of the protein's structure and its interactions with ligands. The findings indicate that both komaroviquinone and brussonol exhibit higher binding affinities compared to mefloquine. This study provides valuable insights into the molecular interactions of these compounds, highlighting their potential for further development as antiviral agents against SARS-CoV-2.

Mechanisms governing bystander activation of T cells.

The immune system is endowed with the capacity to distinguish between self and non-self, so-called immune tolerance or "consciousness of the immune system." This type of awareness is designed to achieve host protection by eliminating cells expressing a wide range of non-self antigens including microbial-derived peptides. Such a successful immune response is associated with the secretion of a whole spectrum of soluble mediators, e.g., cytokines and chemokines, which not only contribute to the clearance of infected host cells but also activate T cells that are not specific to the original cognate antigen. This kind of non-specific T-cell activation is called "bystander activation." Although it is well-established that this phenomenon is cytokine-dependent, there is evidence in the literature showing the involvement of peptide/MHC recognition depending on the type of T-cell subset (naive vs. memory). Here, we will summarize our current understanding of the mechanism(s) of bystander T-cell activation as well as its biological significance in a wide range of diseases including microbial infections, cancer, auto- and alloimmunity, and chronic inflammatory diseases such as atherosclerosis.

Transcriptional profiling of zebrafish intestines identifies macrophages as host cells for human norovirus infection.

Human noroviruses (HuNoVs) are a major cause of diarrheal disease, yet critical aspects of their biology, including cellular tropism, remain unclear. Although research has traditionally focused on the intestinal epithelium, the hypothesis that HuNoV infects macrophages has been recurrently discussed and is investigated here using a zebrafish larval model. Through single-cell RNA sequencing of dissected zebrafish intestines, we unbiasedly identified macrophages as host cells for HuNoV replication, with all three open reading frames mapped to individual macrophages. Notably, HuNoV preferentially infects actively phagocytosing inflammatory macrophages. HuNoV capsid proteins and double-stranded RNA colocalized within intestinal macrophages of infected zebrafish larvae, and the negative-strand RNA intermediate was detected within FACS-sorted macrophages. Flow cytometry confirmed viral replication within these macrophages, constituting approximately 23% of HuNoV's host cells. Identifying macrophages as host cells prompts a reevaluation of their role in HuNoV pathogenesis, offering new directions for understanding and controlling this infection.

Designing a simple and efficient phage biocontainment system using the amber suppressor initiator tRNA.

Multidrug-resistant infections are becoming increasingly prevalent worldwide. One of the fastest-emerging alternative and adjuvant therapies being proposed is phage therapy. Naturally isolated phages are used in the vast majority of phage therapy treatments today. Engineered phages are being developed to enhance the effectiveness of phage therapy, but concerns over their potential escape remain a salient issue. To address this problem, we designed a biocontained phage system based on conditional replication using amber stop codon suppression. This system can be easily installed on any natural phage with a known genome sequence. To test the system, we individually mutated the start codons of three essential capsid genes in phage φX174 to the amber stop codon (UAG). These phages were able to efficiently infect host cells expressing the amber initiator tRNA, which suppresses the amber stop codon and initiates translation at TAG stop codons. The amber phage mutants were also able to successfully infect host cells and reduce their population on solid agar and liquid culture but could not produce infectious particles in the absence of the amber initiator tRNA or complementing capsid gene. We did not detect any growth-inhibiting effects on E. coli strains known to lack a receptor for φX174 and we showed that engineered phages have a limited propensity for reversion. The approach outlined here may be useful to control engineered phage replication in both the lab and clinic.

T4Seeker: a hybrid model for type IV secretion effectors identification

BackgroundThe type IV secretion system is widely present in various bacteria, such as Salmonella, Escherichia coli, and Helicobacter pylori. These bacteria use the type IV secretion system to secrete type IV secretion effectors, infect host cells, and disrupt or modulate the communication pathways. In this study, type III and type VI secretion effectors were used as negative samples to train a robust model.ResultsThe area under the curve of T4Seeker on the validation and independent test sets were 0.947 and 0.970, respectively, demonstrating the strong predictive capacity and robustness of T4Seeker. After comparing with the classic and state-of-the-art T4SE identification models, we found that T4Seeker, which is based on traditional features and large language model features, had a higher predictive ability.ConclusionThe T4Seeker proposed in this study demonstrates superior performance in the field of T4SEs prediction. By integrating features at multiple levels, it achieves higher predictive accuracy and strong generalization capability, providing an effective tool for future T4SE research.

Dynamic metabolic modeling of ATP allocation during viral infection.

Viral pathogens, like SARS-CoV-2, hijack the host's macromolecular production machinery, imposing an energetic burden that is distributed across cellular metabolism. To explore the dynamic metabolic tension between the host's survival and viral replication, we developed a computational framework that uses genome-scale models to perform dynamic Flux Balance Analysis of human cell metabolism during virus infections. Relative to previous models, our framework addresses the physiology of viral infections of non-proliferating host cells through two new features. First, by incorporating the lipid content of SARS-CoV-2 biomass, we discovered activation of previously overlooked pathways giving rise to new predictions of possible drug targets. Furthermore, we introduce a dynamic model that simulates the partitioning of resources between the virus and the host cell, capturing the extent to which the competition depletes the human cells from essential ATP. By incorporating viral dynamics into our COMETS framework for spatio-temporal modeling of metabolism, we provide a mechanistic, dynamic and generalizable starting point for bridging systems biology modeling with viral pathogenesis. This framework could be extended to broadly incorporate phage dynamics in microbial systems and ecosystems.

Fc-FcγR interactions during infections: From neutralizing antibodies to antibody-dependent enhancement.

Advances in antibody technologies have resulted in the development of potent antibody-based therapeutics with proven clinical efficacy against infectious diseases. Several monoclonal antibodies (mAbs), mainly against viruses such as SARS-CoV-2, HIV-1, Ebola virus, influenza virus, and hepatitis B virus, are currently undergoing clinical testing or are already in use. Although these mAbs exhibit potent neutralizing activity that effectively blocks host cell infection, their antiviral activity results not only from Fab-mediated virus neutralization, but also from the protective effector functions mediated through the interaction of their Fc domains with Fcγ receptors (FcγRs) on effector leukocytes. Fc-FcγR interactions confer pleiotropic protective activities, including the clearance of opsonized virions and infected cells, as well as the induction of antiviral T-cell responses. However, excessive or inappropriate activation of specific FcγR pathways can lead to disease enhancement and exacerbated pathology, as seen in the context of dengue virus infections. A comprehensive understanding of the diversity of Fc effector functions during infection has guided the development of engineered antiviral antibodies optimized for maximal effector activity, as well as the design of targeted therapeutic approaches to prevent antibody-dependent enhancement of disease.

Facilitating and restraining virus infection using cell-attachable soluble viral receptors

SARS-CoV-2 uses the receptor binding domain (RBD) of its spike protein to recognize and infect host cells by binding to the cell surface receptor angiotensin converting enzyme 2 (ACE2). The ACE2 receptor is composed of peptidase domain (PD), collectrin-like domain, transmembrane domain, and short cytoplasmic domain, and may exist as a dimer on cell surface. The RBD binding site is located atop of the ACE2 PD, but the involvement of other domains in virus infection is uncertain. We found that the ACE2 PD alone, whether anchored to cell membrane via a glycosylphosphatidylinositol anchor or attached to another surface protein, is fully functional as a receptor for spike-mediated cell fusion and virus infection. However, for ACE2 to function as the viral receptor, the RBD binding site must be positioned in close proximity to the cell membrane. Elevating the surface height of ACE2 using long and rigid protein spacers reduces or eliminates cell fusion and virus infection. Moreover, we found that the RBD-targeting neutralizing antibodies, nanobodies, and de novo designed miniprotein binders, when present on cell surface, also act as viral receptors, facilitating cell fusion and virus infection. Our data demonstrate that RBD binding and close membrane proximity are essential properties for a receptor to effectively mediate SARS-CoV-2 infection. Importantly, we show that soluble RBD-binders can be engineered to make cells either susceptible or resistant to virus infection, which has significant implications for antiviral therapy and various virus-mediated applications.

Leishmania protein KMP-11 modulates cholesterol transport and membrane fluidity to facilitate host cell invasion

The first step of successful infection by any intracellular pathogen relies on its ability to invade its host cell membrane. However, the detailed structural and molecular understanding underlying lipid membrane modification during pathogenic invasion remains unclear. In this study, we show that a specific Leishmania donovani (LD) protein, KMP-11, forms oligomers that bridge LD and host macrophage (MΦ) membranes. This KMP-11 induced interaction between LD and MΦ depends on the variations in cholesterol (CHOL) and ergosterol (ERG) contents in their respective membranes. These variations are crucial for the subsequent steps of invasion, including (a) the initial attachment, (b) CHOL transport from MΦ to LD, and (c) detachment of LD from the initial point of contact through a liquid ordered (Lo) to liquid disordered (Ld) membrane-phase transition. To validate the importance of KMP-11, we generate KMP-11 depleted LD, which failed to attach and invade host MΦ. Through tryptophan-scanning mutagenesis and synthesized peptides, we develop a generalized mathematical model, which demonstrates that the hydrophobic moment and the symmetry sequence code at the membrane interacting protein domain are key factors in facilitating the membrane phase transition and, consequently, the host cell infection process by Leishmania parasites.

Identification of interaction partners of outer inflammatory protein A: Computational and experimental insights into how Helicobacter pylori infects host cells

Outer membrane proteins (OMPs) play a key role in facilitating the survival of Helicobacter pylori within the gastric tissue by mediating adherence. Among these proteins, Outer inflammatory protein A (OipA) is a critical factor in H. pylori colonization of the host gastric epithelial cell surface. While the role of OipA in H. pylori attachment and its association with clinical outcomes have been established, the structural mechanisms underlying OipA’s action in adherence to gastric epithelial cells remain limited. Our study employed experimental and computational approaches to investigate the interaction partners of OipA on the gastric epithelial cell surface. Initially, we conducted a proteomic analysis using a pull-down assay with recombinant OipA and gastric epithelial cell membrane proteins to identify the OipA interactome. This analysis revealed 704 unique proteins that interacted with OipA. We subsequently analyzed 16 of these OipA partners using molecular modeling tools. Among these 16 partners, we highlight three human proteins, namely Hepatocyte growth factor (HGF), Mesenchymal epithelial transition factor receptor (Met), and Adhesion G Protein-Coupled Receptor B1 (AGRB1) that could play a role in H. pylori adherence to the gastric epithelial cell surface with OipA. Collectively, these findings reveal novel host interactions mediated by OipA, suggesting their potential as therapeutic targets for combating H. pylori infection.

Type-I IFNs induce GBPs and lysosomal defense in hepatocytes to control malaria.

Plasmodium parasites undergo development and replication within the hepatocytes before infecting the erythrocytes and initiating clinical malaria. Although type-I interferons (IFNs) are known to hinder Plasmodium infection within the liver, the underlying mechanisms remain unclear. Here, we describe two IFN-I-driven hepatocyte antimicrobial programs controlling liver-stage malaria. First, oxidative defense by NADPH oxidases 2 and 4 triggers a pathway of lysosomal fusion with the parasitophorous vacuole (PV) to help clear Plasmodium . Second, guanylate-binding protein (GBP) 1 disruption of the PV activates caspase-1 inflammasome, inducing pyroptosis to remove the infected host cells. Remarkably, both human and mouse hepatocytes enlist these cell-autonomous immune programs to eliminate Plasmodium ; their pharmacologic or genetic inhibition led to profound malarial susceptibility, and are essential in vivo . In addition to identifying the IFN-I-mediated cell-autonomous immune circuits controlling Plasmodium infection in the hepatocytes, this study extends our understanding of how non-immune cells are integral to protective immunity against malaria.

The host cells suppress the proliferation of pseudorabies virus by regulating the PI3K/Akt/mTOR pathway.

PRV, known for its neurotropic properties, is capable of inducing severe neuronal damage. Our study discovered that following PRV infection, the expression of MMP2 was upregulated, leading to the degradation of ZO-1. Furthermore, upon PRV infection in the host, the promoter of TIMP1 is significantly activated, resulting in a significant increase in TIMP1 protein levels. This upregulation of TIMP1 inhibits the proliferation of PRV through the PI3K/Akt signaling pathway. This study elucidated the mechanism through which PRV, including the PRV XJ delgE/gI/TK strains, compromises the blood-brain barrier and identifies the antiviral response characterized by the activation of the PI3K/Akt signaling pathway within infected host cells. These findings provide potential therapeutic targets for the clinical management and treatment of PRV.

Antibodies as Promising Molecules to Block Spike Glycoprotein from SARS-CoV2

Abstract: COVID-19 is caused by a new SARS-CoV-2 virus from the Coronaviridae family, responsible for developing severe acute respiratory syndrome (SARS-CoV). Its emergence in 2019 presented several challenges to global health. The virus can infect host cells using the ACE2 receptor, and after infection, several clinical manifestations appear, ranging from mild symptoms to death. One approach that has shown promising results is convalescent plasma since convalescent plasma (CP) consists of a concentrate of immunoglobulins in the blood of patients recently infected with the coronavirus for those currently infected. Therefore, this therapy uses antibodies in the plasma to neutralize the virus. This literature review aims to evaluate the efficacy of CP therapy in symptomatic COVID-19 patients by analyzing clinical trials and studies published between 2020 and 2023. Therefore, the search identified studies in which the majority reported the efficacy of using convalescent plasma to treat and alleviate the clinical condition of patients diagnosed with COVID-19. Important factors influence the efficacy of CP therapy, such as the timing of administration and the severity of the disease. Early administration in mild and moderate cases has shown promise. Furthermore, molecular docking analysis suggests that antibodies can cause conformational changes in the SARS-CoV-2 spike protein, raising the hypothesis that using CP may interfere with viral entry into host cells.

Hypermigration of macrophages through the concerted action of GRA effectors on NF-κB/p38 signaling and host chromatin accessibility potentiates Toxoplasma dissemination.

Intracellular pathogens can hijack the cellular functions of infected host cells to their advantage, for example, for intracellular survival and dissemination. However, how microbes orchestrate the hijacking of complex cellular processes, such as host cell migration, remains poorly understood. As such, the common parasite Toxoplasma gondii actively invades the immune cells of humans and other vertebrates and modifies their migratory properties. Here, we show that the concerted action of a number of secreted effector proteins from the parasite, principally GRA15 and GRA24, acts on host cell signaling pathways to activate chemotaxis. Furthermore, the protein effector GRA28 selectively acted on chromatin accessibility in the host cell nucleus to selectively boost host gene expression. The joint activities of GRA effectors culminated in pro-migratory signaling within the infected phagocyte. We provide a molecular framework delineating how T. gondii can orchestrate a complex biological phenotype, such as the migratory activation of phagocytes to boost dissemination.

Deciphering Pathways and Thermodynamics of Protein Assembly Using Native Mass Spectrometry.

Protein oligomerization regulates many critical physiological processes, and its dysregulation can contribute to dysfunction and diseases. Elucidating the assembly pathways and quantifying their underlying thermodynamic and kinetic parameters are crucial for a comprehensive understanding of biological processes and for advancing therapeutics targeting abnormal protein oligomerization. Established binding assays, with limited mass precision, often rely on simplified models for data interpretation. In contrast, high-resolution native mass spectrometry (nMS) can directly determine the stoichiometry of biomolecular complexes in vitro. However, quantification is hindered by the fact that the relative abundances of gas-phase ions generally do not reflect solution concentrations due to nonuniform response factors. Recently, slow mixing mode (SLOMO)-nMS, which can quantify the relative response factors of interacting species, has been demonstrated to reliably measure the affinity (Kd) of binary biomolecular complexes. Here, we introduce an extended form of SLOMO-nMS that enables simultaneous quantification of the thermodynamics in multistep association reactions. Application of this method to homo-oligomerization of concanavalin A and insulin confirmed the reliability of the assay and uncovered details about the assembly processes that had previously resisted elucidation. Results acquired using SLOMO-nMS implemented with charge detection shed new light on the binding of recombinant human angiotensin-converting enzyme 2 and the SARS-CoV-2 spike protein. Importantly, new assembly pathways were uncovered, and the affinities of these interactions, which regulate host cell infection, were quantified. Together, these findings highlight the tremendous potential of SLOMO-nMS to accelerate the characterization of protein assembly pathways and thermodynamics and, in so doing, enhance fundamental biological understanding and facilitate therapeutic development. https://orcid.org/0000-0002-3389-7112.

PlzR regulates type IV pili assembly in Pseudomonas aeruginosa via PilZ binding

Type IV pili (T4P) are thin, flexible filaments exposed on the cell surface of gram-negative bacteria and are involved in pathogenesis-related processes, including cell adsorption, biofilm formation, and twitching motility. Bacteriophages often use these filaments as receptors to infect host cells. Here, we describe the identification of a protein that inhibits T4P assembly in Pseudomonas aeruginosa, discovered during a screen for host factors influencing phage infection. We show that expression of PA2560 (renamed PlzR) in P. aeruginosa inhibits adsorption of T4P-dependent phages. PlzR does this by directly binding the T4P chaperone PilZ, which in turn regulates the ATPase PilB and results in disturbed T4P assembly. As the plzR promoter is induced by cyclic di-GMP, PlzR might play a role in coupling T4P function to levels of this second messenger.