Novel "GaEl Antigenic Patches" Identified by a "Reverse Epitomics" Approach to Design Multipatch Vaccines against NIPAH Infection, a Silent Threat to Global Human Health.

Nipah virus (NiV) is a zoonotic virus that causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. Several NiV outbreaks have been reported since 1999 with nearly annual occurrences in Bangladesh. The outbreaks had high mortality rates ranging from 40 to 90%. No specific vaccine has yet been reported against NiV. Recently, several vaccine candidates and different designs of vaccines composed of epitopes against NiV were proposed. Most of the vaccines target single protein or protein complex subunits of the pathogen. The multiepitope vaccines proposed also cover a largely limited number of epitopes, and hence, their efficiency is still uncertain. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have utilized the "reverse epitomics" approach ("overlapping-epitope-clusters-to-patches" method) to identify "antigenic patches" (Ag-Patches) and utilize them as immunogenic composition for multipatch vaccine (MPV) design. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties, and interaction with Toll-like receptor 3 ectodomain. In total, 30 CTL (cytotoxic T lymphocyte) and 27 HTL (helper T lymphocyte) antigenic patches were identified from the entire NiV proteome based on the clusters of overlapping epitopes. These identified Ag-Patches cover a total of discrete 362 CTL and 414 HTL epitopes from the entire proteome of NiV. The antigenic patches were utilized as immunogenic composition for the design of two CTL and two HTL multipatch vaccines. The 57 antigenic patches utilized here cover 776 overlapping epitopes targeting 52 different HLA class I and II alleles, providing a global ethnically distributed human population coverage of 99.71%. Such large number of epitope coverage resulting in large human population coverage cannot be reached with single-protein/subunit or multiepitope based vaccines. The reported antigenic patches also provide potential immunogenic composition for early detection diagnostic kits for NiV infection. Further, all the MPVs and Toll-like receptor ectodomain complexes show a stable nature of molecular interaction with numerous hydrogen bonds, salt bridges, and nonbounded contact formation and acceptable root mean square deviation and fluctuation. The cDNA analysis shows a favorable large-scale expression of the MPV constructs in a human cell line. By utilizing the novel "reverse epitomics" approach, highly immunogenic novel "GaEl antigenic patches" (GaEl Ag-Patches), a synonym term for "antigenic patches", were identified and utilized as immunogenic composition to design four MPVs against NiV. We conclude that the novel multipatch vaccines are potential candidates to combat NiV, with greater effectiveness, high specificity, and large human population coverage worldwide.

Exploring the structural basis to develop efficient multi-epitope vaccines displaying interaction with HLA and TAP and TLR3 molecules to prevent NIPAH infection, a global threat to human health.

Nipah virus (NiV) is an emerging zoonotic virus that caused several serious outbreaks in the south asian region with high mortality rates ranging from 40 to 90% since 2001. NiV infection causes lethal encephalitis and respiratory disease with the symptom of endothelial cell-cell fusion. No specific and effective vaccine has yet been reported against NiV. To address the urgent need for a specific and effective vaccine against NiV infection, in the present study, we have designed two Multi-Epitope Vaccines (MEVs) composed of 33 Cytotoxic T lymphocyte (CTL) epitopes and 38 Helper T lymphocyte (HTL) epitopes. Out of those CTL and HTL combined 71 epitopes, 61 novel epitopes targeting nine different NiV proteins were not used before for vaccine design. Codon optimization for the cDNA of both the designed MEVs might ensure high expression potential in the human cell line as stable proteins. Both MEVs carry potential B cell linear epitope overlapping regions, B cell discontinuous epitopes as well as IFN-γ inducing epitopes. Additional criteria such as sequence consensus amongst CTL, HTL and B Cell epitopes was implemented for the design of final constructs constituting MEVs. Hence, the designed MEVs carry the potential to elicit cell-mediated as well as humoral immune response. Selected overlapping CTL and HTL epitopes were validated for their stable molecular interactions with HLA class I and II alleles and in case of CTL epitopes with human Transporter Associated with antigen Processing (TAP) cavity. The structure based epitope cross validation for interaction with TAP cavity was used as another criteria choosing final epitopes for NiV MEVs. Finally, human Beta-defensin 2 and Beta-defensin 3 were used as adjuvants to enhance the immune response of both the MEVs. Molecular dynamics simulation studies of MEVs-TLR3 ectodomain (Human Toll-Like Receptor 3) complex indicated the stable molecular interaction. We conclude that the MEVs designed and in silico validated here could be highly potential vaccine candidates to combat NiV infections, with great effectiveness, high specificity and large human population coverage worldwide.

Integrative structural analysis of the type III secretion system needle complex from Shigella flexneri.

The type III secretion system (T3SS) is a large, transmembrane protein machinery used by various pathogenic gram-negative bacteria to transport virulence factors into the host cell during infection. Understanding the structure of T3SSs is crucial for future developments of therapeutics that could target this system. However, much of the knowledge about the structure of T3SS is available only for Salmonella, and it is unclear how this large assembly is conserved across species. Here, we combined cryo-electron microscopy, cross-linking mass spectrometry, and integrative modeling to determine the structure of the T3SS needle complex from Shigella flexneri. We show that the Shigella T3SS exhibits unique features distinguishing it from other structurally characterized T3SSs. The secretin pore complex adopts a new fold of its C-terminal S domain and the pilotin MxiM[SctG] locates around the outer surface of the pore. The export apparatus structure exhibits a conserved pseudohelical arrangement but includes the N-terminal domain of the SpaS[SctU] subunit, which was not present in any of the previously published virulence-related T3SS structures. Similar to other T3SSs, however, the apparatus is anchored within the needle complex by a network of flexible linkers that either adjust conformation to connect to equivalent patches on the secretin oligomer or bind distinct surface patches at the same height of the export apparatus. The conserved and unique features delineated by our analysis highlight the necessity to analyze T3SS in a species-specific manner, in order to fully understand the underlying molecular mechanisms of these systems. The structure of the type III secretion system from Shigella flexneri delineates conserved and unique features, which could be used for the development of broad-range therapeutics.

Determination of optical constants of thin films in the EUV.

The determination of fundamental optical parameters is essential for the development of new optical elements such as mirrors, gratings, or photomasks. Especially in the extreme ultraviolet (EUV) and soft x-ray spectral range, the existing databases for the refractive indices of many materials and compositions are insufficient or are a mixture of experimentally measured and calculated values from atomic scattering factors. Since the physical properties of bulk materials and thin films with thicknesses in the nanometer range are not identical, measurements need to be performed on thin layers. In this study we demonstrate how optical constants of various thin film samples on a bulk substrate can be determined from reflection measurements in the EUV photon energy range from 62 eV to 124 eV. Thin films with thickness of 20 nm to 50 nm of pure Mo, Ni, Pt, Ru, Ta, and Te and different compositions of NixAlx, PtTe, PtxMo, RuxTax, Ru3Re, Ru2W, and TaTeN were prepared by DC magnetron sputtering and measured using EUV reflectometry. The determination optical constants of the different materials are discussed and compared to existing tabulated values.

Computationally validated SARS-CoV-2 CTL and HTL Multi-Patch vaccines, designed by reverse epitomics approach, show potential to cover large ethnically distributed human population worldwide.

The SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is responsible for the COVID-19 outbreak. The highly contagious COVID-19 disease has spread to 216 countries in less than six months. Though several vaccine candidates are being claimed, an effective vaccine is yet to come. A novel reverse epitomics approach, 'overlapping-epitope-clusters-to-patches' method is utilized to identify the antigenic regions from the SARS-CoV-2 proteome. These antigenic regions are named as 'Ag-Patch or Ag-Patches', for Antigenic Patch or Patches. The identification of Ag-Patches is based on the clusters of overlapping epitopes rising from SARS-CoV-2 proteins. Further, we have utilized the identified Ag-Patches to design Multi-Patch Vaccines (MPVs), proposing a novel method for the vaccine design. The designed MPVs were analyzed for immunologically crucial parameters, physiochemical properties and cDNA constructs. We identified 73 CTL (Cytotoxic T-Lymphocyte) and 49 HTL (Helper T-Lymphocyte) novel Ag-Patches from the proteome of SARS-CoV-2. The identified Ag-Patches utilized to design MPVs cover 768 overlapping epitopes targeting 55 different HLA alleles leading to 99.98% of world human population coverage. The MPVs and Toll-Like Receptor ectodomain complex shows stable complex formation tendency. Further, the cDNA analysis favors high expression of the MPVs constructs in a human cell line. We identified highly immunogenic novel Ag-Patches from the entire proteome of SARS CoV-2 by a novel reverse epitomics approach and utilized them to design MPVs. We conclude that the novel MPVs could be a highly potential novel approach to combat SARS-CoV-2, with greater effectiveness, high specificity and large human population coverage worldwide. Communicated by Ramaswamy H. Sarma.

Vaccines Targeting Numerous Coronavirus Antigens, Ensuring Broader Global Population Coverage: Multi-epitope and Multi-patch Vaccines.

Coronaviruses are causative agents of different zoonosis including SARS, MERS, or COVID-19 in humans. The high transmission rate of coronaviruses, the time-consuming development of efficient anti-infectives and vaccines, the possible evolutionary adaptation of the virus to conventional vaccines, and the challenge to cover broad human population worldwide are the major reasons that made it challenging to avoid coronaviruses outbreaks. Although, a plethora of different approaches are being followed to design and develop vaccines against coronaviruses, most of them target subunits, full-length single, or only a very limited number of proteins. Vaccine targeting multiple proteins or even the entire proteome of the coronavirus is yet to come. In the present chapter, we will be discussing multi-epitope vaccine (MEV) and multi-patch vaccine (MPV) approaches to design and develop efficient and sustainably successful strategies against coronaviruses. MEV and MPV utilize highly conserved, potentially immunogenic epitopes and antigenic patches, respectively, and hence they have the potential to target large number of coronavirus proteins or even its entire proteome, allowing us to combat the challenge of its evolutionary adaptation. In addition, the large number of human leukocyte antigen (HLA) alleles targeted by the chosen specific epitopes enables MEV and MPV to cover broader global population.

Flagellin lysine methyltransferase FliB catalyzes a [4Fe-4S] mediated methyl transfer reaction.

The methyltransferase FliB posttranslationally modifies surface-exposed ɛ-N-lysine residues of flagellin, the protomer of the flagellar filament in Salmonella enterica (S. enterica). Flagellin methylation, reported originally in 1959, was recently shown to enhance host cell adhesion and invasion by increasing the flagellar hydrophobicity. The role of FliB in this process, however, remained enigmatic. In this study, we investigated the properties and mechanisms of FliB from S. enterica in vivo and in vitro. We show that FliB is an S-adenosylmethionine (SAM) dependent methyltransferase, forming a membrane associated oligomer that modifies flagellin in the bacterial cytosol. Using X-band electron paramagnetic resonance (EPR) spectroscopy, zero-field 57Fe Mössbauer spectroscopy, methylation assays and chromatography coupled mass spectrometry (MS) analysis, we further found that FliB contains an oxygen sensitive [4Fe-4S] cluster that is essential for the methyl transfer reaction and might mediate a radical mechanism. Our data indicate that the [4Fe-4S] cluster is coordinated by a cysteine rich motif in FliB that is highly conserved among multiple genera of the Enterobacteriaceae family.

Helical reconstruction of Salmonella and Shigella needle filaments attached to type 3 basal bodies.

Gram-negative pathogens evolved a syringe-like nanomachine, termed type 3 secretion system, to deliver protein effectors into the cytoplasm of host cells. An essential component of this system is a long helical needle filament that protrudes from the bacterial surface and connects the cytoplasms of the bacterium and the eukaryotic cell. Previous structural research was predominantly focused on reconstituted type 3 needle filaments, which lacked the biological context. In this work we introduce a facile procedure to obtain high-resolution cryo-EM structure of needle filaments attached to the basal body of type 3 secretion systems. We validate our approach by solving the structure of Salmonella PrgI filament and demonstrate its utility by obtaining the first high-resolution cryo-EM reconstruction of Shigella MxiH filament. Our work paves the way to systematic structural characterization of attached type 3 needle filaments in the context of mutagenesis studies, protein structural evolution and drug development.

Novel Method for Quantifying AhR-Ligand Binding Affinities Using Microscale Thermophoresis.

The aryl hydrocarbon receptor (AhR) is a highly conserved cellular sensor of a variety of environmental pollutants and dietary-, cell- and microbiota-derived metabolites with important roles in fundamental biological processes. Deregulation of the AhR pathway is implicated in several diseases, including autoimmune diseases and cancer, rendering AhR a promising target for drug development and host-directed therapy. The pharmacological intervention of AhR processes requires detailed information about the ligand binding properties to allow specific targeting of a particular signaling process without affecting the remaining. Here, we present a novel microscale thermophoresis-based approach to monitoring the binding of purified recombinant human AhR to its natural ligands in a cell-free system. This approach facilitates a precise identification and characterization of unknown AhR ligands and represents a screening strategy for the discovery of potential selective AhR modulators.

Structural Basis for Designing Multiepitope Vaccines Against COVID-19 Infection: In Silico Vaccine Design and Validation.

BACKGROUND: The novel coronavirus disease (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has led to the ongoing 2019-2020 pandemic. SARS-CoV-2 is a positive-sense single-stranded RNA coronavirus. Effective countermeasures against SARS-CoV-2 infection require the design and development of specific and effective vaccine candidates. OBJECTIVE: To address the urgent need for a SARS-CoV-2 vaccine, in the present study, we designed and validated one cytotoxic T lymphocyte (CTL) and one helper T lymphocyte (HTL) multi-epitope vaccine (MEV) against SARS-CoV-2 using various in silico methods. METHODS: Both designed MEVs are composed of CTL and HTL epitopes screened from 11 Open Reading Frame (ORF), structural and nonstructural proteins of the SARS-CoV-2 proteome. Both MEVs also carry potential B-cell linear and discontinuous epitopes as well as interferon gamma-inducing epitopes. To enhance the immune response of our vaccine design, truncated (residues 10-153) Onchocerca volvulus activation-associated secreted protein-1 was used as an adjuvant at the N termini of both MEVs. The tertiary models for both the designed MEVs were generated, refined, and further analyzed for stable molecular interaction with toll-like receptor 3. Codon-biased complementary DNA (cDNA) was generated for both MEVs and analyzed in silico for high level expression in a mammalian (human) host cell line. RESULTS: In the present study, we screened and shortlisted 38 CTL, 33 HTL, and 12 B cell epitopes from the 11 ORF protein sequences of the SARS-CoV-2 proteome. Moreover, the molecular interactions of the screened epitopes with their respective human leukocyte antigen allele binders and the transporter associated with antigen processing (TAP) complex were positively validated. The shortlisted screened epitopes were utilized to design two novel MEVs against SARS-CoV-2. Further molecular models of both MEVs were prepared, and their stable molecular interactions with toll-like receptor 3 were positively validated. The codon-optimized cDNAs of both MEVs were also positively analyzed for high levels of overexpression in a human cell line. CONCLUSIONS: The present study is highly significant in terms of the molecular design of prospective CTL and HTL vaccines against SARS-CoV-2 infection with potential to elicit cellular and humoral immune responses. The epitopes of the designed MEVs are predicted to cover the large human population worldwide (96.10%). Hence, both designed MEVs could be tried in vivo as potential vaccine candidates against SARS-CoV-2.

Methylation of Salmonella Typhimurium flagella promotes bacterial adhesion and host cell invasion.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Cryo-EM structure of the Shigella type III needle complex.

The Type III Secretion Systems (T3SS) needle complex is a conserved syringe-shaped protein translocation nanomachine with a mass of about 3.5 MDa essential for the survival and virulence of many Gram-negative bacterial pathogens. This system is composed of a membrane-embedded basal body and an extracellular needle that deliver effector proteins into host cells. High-resolution structures of the T3SS from different organisms and infection stages are needed to understand the underlying molecular mechanisms of effector translocation. Here, we present the cryo-electron microscopy structure of the isolated Shigella T3SS needle complex. The inner membrane (IM) region of the basal body adopts 24-fold rotational symmetry and forms a channel system that connects the bacterial periplasm with the export apparatus cage. The secretin oligomer adopts a heterogeneous architecture with 16- and 15-fold cyclic symmetry in the periplasmic N-terminal connector and C-terminal outer membrane ring, respectively. Two out of three IM subunits bind the secretin connector via a ß-sheet augmentation. The cryo-EM map also reveals the helical architecture of the export apparatus core, the inner rod, the needle and their intervening interfaces.

Host monitoring of quorum sensing during Pseudomonas aeruginosa infection.

Pseudomonas aeruginosa rapidly adapts to altered conditions by quorum sensing (QS), a communication system that it uses to collectively modify its behavior through the production, release, and detection of signaling molecules. QS molecules can also be sensed by hosts, although the respective receptors and signaling pathways are poorly understood. We describe a pattern of regulation in the host by the aryl hydrocarbon receptor (AhR) that is critically dependent on qualitative and quantitative sensing of P. aeruginosa quorum. QS molecules bind to AhR and distinctly modulate its activity. This is mirrored upon infection with P. aeruginosa collected from diverse growth stages and with QS mutants. We propose that by spying on bacterial quorum, AhR acts as a major sensor of infection dynamics, capable of orchestrating host defense according to the status quo of infection.

Role of flagellar hydrogen bonding in Salmonella motility and flagellar polymorphic transition.

Bacterial flagellar filaments are assembled by tens of thousands flagellin subunits, forming 11 helically arranged protofilaments. Each protofilament can take either of the two bistable forms L-type or R-type, having slightly different conformations and inter-protofilaments interactions. By mixing different ratios of L-type and R-type protofilaments, flagella adopt multiple filament polymorphs and promote bacterial motility. In this study, we investigated the hydrogen bonding networks at the flagellin crystal packing interface in Salmonella enterica serovar typhimurium (S. typhimurium) by site-directed mutagenesis of each hydrogen bonded residue. We identified three flagellin mutants D108A, N133A and D152A that were non-motile despite their fully assembled flagella. Mutants D108A and D152A trapped their flagellar filament into inflexible right-handed polymorphs, which resemble the previously predicted 3L/8R and 4L/7R helical forms in Calladine's model but have never been reported in vivo. Mutant N133A produces floppy flagella that transform flagellar polymorphs in a disordered manner, preventing the formation of flagellar bundles. Further, we found that the hydrogen bonding interactions around these residues are conserved and coupled to flagellin L/R transition. Therefore, we demonstrate that the hydrogen bonding networks formed around flagellin residues D108, N133 and D152 greatly contribute to flagellar bending, flexibility, polymorphisms and bacterial motility.

Structural analysis of ligand-bound states of the Salmonella type III secretion system ATPase InvC.

Translocation of virulence effector proteins through the type III secretion system (T3SS) is essential for the virulence of many medically relevant Gram-negative bacteria. The T3SS ATPases are conserved components that specifically recognize chaperone-effector complexes and energize effector secretion through the system. It is thought that functional T3SS ATPases assemble into a cylindrical structure maintained by their N-terminal domains. Using size-exclusion chromatography coupled to multi-angle light scattering and native mass spectrometry, we show that in the absence of the N-terminal oligomerization domain the Salmonella T3SS ATPase InvC can form monomers and dimers in solution. We also present for the first time a 2.05 å resolution crystal structure of InvC lacking the oligomerization domain (InvCΔ79) and map the amino acids suggested for ATPase intersubunit interaction, binding to other T3SS proteins and chaperone-effector recognition. Furthermore, we validate the InvC ATP-binding site by co-crystallization of InvCΔ79 with ATPγS (2.65 å) and ADP (2.80 å). Upon ATP-analogue recognition, these structures reveal remodeling of the ATP-binding site and conformational changes of two loops located outside of the catalytic site. Both loops face the central pore of the predicted InvC cylinder and are essential for the function of the T3SS ATPase. Our results present a fine functional and structural correlation of InvC and provide further details of the homo-oligomerization process and ATP-dependent conformational changes underlying the T3SS ATPase activity.

Molecular Organization of Soluble Type III Secretion System Sorting Platform Complexes.

Many medically relevant Gram-negative bacteria use the type III secretion system (T3SS) to translocate effector proteins into the host for their invasion and intracellular survival. A multi-protein complex located at the cytosolic interface of the T3SS is proposed to act as a sorting platform by selecting and targeting substrates for secretion through the system. However, the precise stoichiometry and 3D organization of the sorting platform components are unknown. Here we reconstitute soluble complexes of the Salmonella Typhimurium sorting platform proteins including the ATPase InvC, the regulator OrgB, the protein SpaO and a recently identified subunit SpaOC, which we show to be essential for the solubility of SpaO. We establish domain-domain interactions, determine for the first time the stoichiometry of each subunit within the complexes by native mass spectrometry and gain insight into their organization using small-angle X-ray scattering. Importantly, we find that in solution the assembly of SpaO/SpaOC/OrgB/InvC adopts an extended L-shaped conformation resembling the sorting platform pods seen in in situ cryo-electron tomography, proposing that this complex is the core building block that can be conceivably assembled into higher oligomers to form the T3SS sorting platform. The determined molecular arrangements of the soluble complexes of the sorting platform provide important insights into its architecture and assembly.

cGAS facilitates sensing of extracellular cyclic dinucleotides to activate innate immunity.

Cyclic dinucleotides (CDNs) are important second messenger molecules in prokaryotes and eukaryotes. Within host cells, cytosolic CDNs are detected by STING and alert the host by activating innate immunity characterized by type I interferon (IFN) responses. Extracellular bacteria and dying cells can release CDNs, but sensing of extracellular CDNs (eCDNs) by mammalian cells remains elusive. Here, we report that endocytosis facilitates internalization of eCDNs. The DNA sensor cGAS facilitates sensing of endocytosed CDNs, their perinuclear accumulation, and subsequent STING-dependent release of type I IFN Internalized CDNs bind cGAS directly, leading to its dimerization, and the formation of a cGAS/STING complex, which may activate downstream signaling. Thus, eCDNs comprise microbe- and danger-associated molecular patterns that contribute to host-microbe crosstalk during health and disease.

The U125 insertion device beamline at the Metrology Light Source.

At the Metrology Light Source, an electron storage ring dedicated to metrological applications, the U125 insertion device beamline utilizes undulator radiation for various applications over a broad spectral range. Using a hybrid normal-incidence and grazing-incidence in-vacuum switchable plane-grating monochromator, a spectral region ranging from the near-infrared to soft X-ray is covered. The beamline is dedicated to surface-analytical methods, e.g. ellipsometry, photoelectron spectroscopy or photoemission tomography. The traceability of radiometric quantities, i.e. quantitative determination of the available radiant power (or photon flux), is required for some of these applications to support the metrological aspect of the measurements. In particular, attention is paid to the suppression of unwanted spectral contributions from higher diffraction orders, and to the monitoring of the radiation intensity during the measurements. With the results from the beamline commissioning, an uncertainty budget for all relevant radiometric quantities was established.

Flagellin phase-dependent swimming on epithelial cell surfaces contributes to productive Salmonella gut colonisation.

The flagellum is a sophisticated nanomachine and an important virulence factor of many pathogenic bacteria. Flagellar motility enables directed movements towards host cells in a chemotactic process, and near-surface swimming on cell surfaces is crucial for selection of permissive entry sites. The long external flagellar filament is made of tens of thousands subunits of a single protein, flagellin, and many Salmonella serovars alternate expression of antigenically distinct flagellin proteins, FliC and FljB. However, the role of the different flagellin variants during gut colonisation and host cell invasion remains elusive. Here, we demonstrate that flagella made of different flagellin variants display structural differences and affect Salmonella's swimming behaviour on host cell surfaces. We observed a distinct advantage of bacteria expressing FliC-flagella to identify target sites on host cell surfaces and to invade epithelial cells. FliC-expressing bacteria outcompeted FljB-expressing bacteria for intestinal tissue colonisation in the gastroenteritis and typhoid murine infection models. Intracellular survival and responses of the host immune system were not altered. We conclude that structural properties of flagella modulate the swimming behaviour on host cell surfaces, which facilitates the search for invasion sites and might constitute a general mechanism for productive host cell invasion of flagellated bacteria.