Microbiology: Murder as a solution to promiscuity?

Strain-specific pili enable Vibrio cholerae bacteria to adhere to each other and form aggregates in liquid culture. A new study focuses on strains with less specific, promiscuous pili and suggests a role for contact-dependent bacterial killing in shaping the composition of these aggregates.

Lack of physiological evidence for cytochrome filaments functioning as conduits for extracellular electron transfer.

Extracellular cytochrome filaments are proposed to serve as conduits for long-range extracellular electron transfer. The primary functional physiological evidence has been the reported inhibition of Geobacter sulfurreducens Fe(III) oxide reduction when the gene for the filament-forming cytochrome OmcS is deleted. Here we report that the OmcS-deficient strain from that original report reduces Fe(III) oxide as well as the wild-type, as does a triple mutant in which the genes for the other known filament-forming cytochromes were also deleted. The triple cytochrome mutant displayed filaments with the same 3 nm diameter morphology and conductance as those produced by Escherichia coli heterologously expressing the G. sulfurreducens PilA pilin gene. Fe(III) oxide reduction was inhibited when the pilin gene in cytochrome-deficient mutants was modified to yield poorly conductive 3 nm diameter filaments. The results are consistent with the concept that 3 nm diameter electrically conductive pili (e-pili) are required for G. sulfurreducens long-range extracellular electron transfer. In contrast, rigorous physiological functional evidence is lacking for cytochrome filaments serving as conduits for long-range electron transport. IMPORTANCE: Unraveling microbial extracellular electron transfer mechanisms has profound implications for environmental processes and advancing biological applications. This study on Geobacter sulfurreducens challenges prevailing beliefs on cytochrome filaments as crucial components thought to facilitate long-range electron transport. The discovery of an OmcS-deficient strain's unexpected effectiveness in Fe(III) oxide reduction prompted a reevaluation of the key conduits for extracellular electron transfer. By exploring the impact of genetic modifications on G. sulfurreducens' performance, this research sheds light on the importance of 3-nm diameter electrically conductive pili in Fe(III) oxide reduction. Reassessing these mechanisms is essential for uncovering the true drivers of extracellular electron transfer in microbial systems, offering insights that could revolutionize applications across diverse fields.

Structural mechanisms of Tad pilus assembly and its interaction with an RNA virus.

Caulobacter crescentus Tad (tight adherence) pili, part of the type IV pili family, are crucial for mechanosensing, surface adherence, bacteriophage (phage) adsorption, and cell-cycle regulation. Unlike other type IV pilins, Tad pilins lack the typical globular ß sheet domain responsible for pilus assembly and phage binding. The mechanisms of Tad pilus assembly and its interaction with phage ΦCb5 have been elusive. Using cryo-electron microscopy, we unveiled the Tad pilus assembly mechanism, featuring a unique network of hydrogen bonds at its core. We then identified the Tad pilus binding to the ΦCb5 maturation protein (Mat) through its ß region. Notably, the amino terminus of ΦCb5 Mat is exposed outside the capsid and phage/pilus interface, enabling the attachment of fluorescent and affinity tags. These engineered ΦCb5 virions can be efficiently assembled and purified in Escherichia coli, maintaining infectivity against C. crescentus, which presents promising applications, including RNA delivery and phage display.

Data-driven modelling makes quantitative predictions regarding bacteria surface motility.

In this work, we quantitatively compare computer simulations and existing cell tracking data of P. aeruginosa surface motility in order to analyse the underlying motility mechanism. We present a three dimensional twitching motility model, that simulates the extension, retraction and surface association of individual Type IV Pili (TFP), and is informed by recent experimental observations of TFP. Sensitivity analysis is implemented to minimise the number of model parameters, and quantitative estimates for the remaining parameters are inferred from tracking data by approximate Bayesian computation. We argue that the motility mechanism is highly sensitive to experimental conditions. We predict a TFP retraction speed for the tracking data we study that is in a good agreement with experimental results obtained under very similar conditions. Furthermore, we examine whether estimates for biologically important parameters, whose direct experimental determination is challenging, can be inferred directly from tracking data. One example is the width of the distribution of TFP on the bacteria body. We predict that the TFP are broadly distributed over the bacteria pole in both walking and crawling motility types. Moreover, we identified specific configurations of TFP that lead to transitions between walking and crawling states.

Functional Analysis of the Major Pilin Proteins of Type IV Pili in Streptococcus sanguinis CGMH010.

The pil gene cluster for Type IV pilus (Tfp) biosynthesis is commonly present and highly conserved in Streptococcus sanguinis. Nevertheless, Tfp-mediated twitching motility is less common among strains, and the factors determining twitching activity are not fully understood. Here, we analyzed the functions of three major pilin proteins (PilA1, PilA2, and PilA3) in the assembly and activity of Tfp in motile S. sanguinis CGMH010. Using various recombinant pilA deletion strains, we found that Tfp composed of different PilA proteins varied morphologically and functionally. Among the three PilA proteins, PilA1 was most critical in the assembly of twitching-active Tfp, and recombinant strains expressing motility generated more structured biofilms under constant shearing forces compared to the non-motile recombinant strains. Although PilA1 and PilA3 shared 94% identity, PilA3 could not compensate for the loss of PilA1, suggesting that the nature of PilA proteins plays an essential role in twitching activity. The single deletion of individual pilA genes had little effect on the invasion of host endothelia by S. sanguinis CGMH010. In contrast, the deletion of all three pilA genes or pilT, encoding the retraction ATPase, abolished Tfp-mediated invasion. Tfp- and PilT-dependent invasion were also detected in the non-motile S. sanguinis SK36, and thus, the retraction of Tfp, but not active twitching, was found to be essential for invasion.

Interactions between pili affect the outcome of bacterial competition driven by the type VI secretion system.

The bacterial type VI secretion system (T6SS) is a widespread, kin-discriminatory weapon capable of shaping microbial communities. Due to the system's dependency on contact, cellular interactions can lead to either competition or kin protection. Cell-to-cell contact is often accomplished via surface-exposed type IV pili (T4Ps). In Vibrio cholerae, these T4Ps facilitate specific interactions when the bacteria colonize natural chitinous surfaces. However, it has remained unclear whether and, if so, how these interactions affect the bacterium's T6SS-mediated killing. In this study, we demonstrate that pilus-mediated interactions can be harnessed by T6SS-equipped V. cholerae to kill non-kin cells under liquid growth conditions. We also show that the naturally occurring diversity of pili determines the likelihood of cell-to-cell contact and, consequently, the extent of T6SS-mediated competition. To determine the factors that enable or hinder the T6SS's targeted reduction of competitors carrying pili, we developed a physics-grounded computational model for autoaggregation. Collectively, our research demonstrates that T4Ps involved in cell-to-cell contact can impose a selective burden when V. cholerae encounters non-kin cells that possess an active T6SS. Additionally, our study underscores the significance of T4P diversity in protecting closely related individuals from T6SS attacks through autoaggregation and spatial segregation.

The assembly platform FimD is required to obtain the most stable quaternary structure of type 1 pili.

Type 1 pili are important virulence factors of uropathogenic Escherichia coli that mediate bacterial attachment to epithelial cells in the urinary tract. The pilus rod is comprised of thousands of copies of the main structural subunit FimA and is assembled in vivo by the assembly platform FimD. Although type 1 pilus rods can self-assemble from FimA in vitro, this reaction is slower and produces structures with lower kinetic stability against denaturants compared to in vivo-assembled rods. Our study reveals that FimD-catalysed in vitro-assembled type 1 pilus rods attain a similar stability as pilus rods assembled in vivo. Employing structural, biophysical and biochemical analyses, we show that in vitro assembly reactions lacking FimD produce pilus rods with structural defects, reducing their stability against dissociation. Overall, our results indicate that FimD is not only required for the catalysis of pilus assembly, but also to control the assembly of the most stable quaternary structure.

Recombinational exchange of M-fibril and T-pilus genes generates extensive cell surface diversity in the global group A Streptococcus population.

Among genes present in all group A streptococci (GAS), those encoding M-fibril and T-pilus proteins display the highest levels of sequence diversity, giving rise to the two primary serological typing schemes historically used to define strain. A new genotyping scheme for the pilin adhesin and backbone genes is developed and, when combined with emm typing, provides an account of the global GAS strain population. Cluster analysis based on nucleotide sequence similarity assigns most T-serotypes to discrete pilin backbone sequence clusters, yet the established T-types correspond to only half the clusters. The major pilin adhesin and backbone sequence clusters yield 98 unique combinations, defined as "pilin types." Numerous horizontal transfer events that involve pilin or emm genes generate extensive antigenic and functional diversity on the bacterial cell surface and lead to the emergence of new strains. Inferred pilin genotypes applied to a meta-analysis of global population-based collections of pharyngitis and impetigo isolates reveal highly significant associations between pilin genotypes and GAS infection at distinct ecological niches, consistent with a role for pilin gene products in adaptive evolution. Integration of emm and pilin typing into open-access online tools (pubmlst.org) ensures broad utility for end-users wanting to determine the architecture of M-fibril and T-pilus genes from genome assemblies.IMPORTANCEPrecision in defining the variant forms of infectious agents is critical to understanding their population biology and the epidemiology of associated diseases. Group A Streptococcus (GAS) is a global pathogen that causes a wide range of diseases and displays a highly diverse cell surface due to the antigenic heterogeneity of M-fibril and T-pilus proteins which also act as virulence factors of varied functions. emm genotyping is well-established and highly utilized, but there is no counterpart for pilin genes. A global GAS collection provides the basis for a comprehensive pilin typing scheme, and online tools for determining emm and pilin genotypes are developed. Application of these tools reveals the expansion of structural-functional diversity among GAS via horizontal gene transfer, as evidenced by unique combinations of surface protein genes. Pilin and emm genotype correlations with superficial throat vs skin infection provide new insights on the molecular determinants underlying key ecological and epidemiological trends.

Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

Removal of Pseudomonas type IV pili by a small RNA virus.

The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogen Pseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen's virulence. Using cryo-electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics.

Secondary messenger signalling influences Pseudomonas aeruginosa adaptation to sinus and lung environments.

Pseudomonas aeruginosa is a cause of chronic respiratory tract infections in people with cystic fibrosis (CF), non-CF bronchiectasis, and chronic obstructive pulmonary disease. Prolonged infection allows the accumulation of mutations and horizontal gene transfer, increasing the likelihood of adaptive phenotypic traits. Adaptation is proposed to arise first in bacterial populations colonizing upper airway environments. Here, we model this process using an experimental evolution approach. Pseudomonas aeruginosa PAO1, which is not airway adapted, was serially passaged, separately, in media chemically reflective of upper or lower airway environments. To explore whether the CF environment selects for unique traits, we separately passaged PAO1 in airway-mimicking media with or without CF-specific factors. Our findings demonstrated that all airway environments-sinus and lungs, under CF and non-CF conditions-selected for loss of twitching motility, increased resistance to multiple antibiotic classes, and a hyper-biofilm phenotype. These traits conferred increased airway colonization potential in an in vivo model. CF-like conditions exerted stronger selective pressures, leading to emergence of more pronounced phenotypes. Loss of twitching was associated with mutations in type IV pili genes. Type IV pili mediate surface attachment, twitching, and induction of cAMP signalling. We additionally identified multiple evolutionary routes to increased biofilm formation involving regulation of cyclic-di-GMP signalling. These included the loss of function mutations in bifA and dipA phosphodiesterase genes and activating mutations in the siaA phosphatase. These data highlight that airway environments select for traits associated with sessile lifestyles and suggest upper airway niches support emergence of phenotypes that promote establishment of lung infection.

Transcriptomic insights unveil the crucial roles of cytochromes, NADH, and pili in Ag(I) reduction by Geobacter sulfurreducens.

Silver (Ag) is a pivotal transition metal with applications in multiple industries, necessitating efficient recovery techniques. Despite various proposed methods for silver recovery from wastewaters, challenges persist especially for low concentrations. In this context, bioreduction by bacteria like Geobacter sulfurreducens, offers a promising approach by converting Ag(I) to Ag nanoparticles. To reveal the mechanisms driving microbial Ag(I) reduction, we conducted transcriptional profiling of G. sulfurreducens under Ag(I)-reducing condition. Integrated transcriptomic and protein-protein interaction network analyses identified significant transcriptional shifts, predominantly linked to c-type cytochromes, NADH, and pili. When compared to a pilus-deficient strain, the wild-type strain exhibited distinct cytochrome gene expressions, implying specialized functional roles. Additionally, despite a down-regulation in NADH dehydrogenase genes, we observed up-regulation of specific downstream cytochrome genes, highlighting NADH's potential role as an electron donor in the Ag(I) reduction process. Intriguingly, our findings also highlight the significant influence of pili on the morphology of the resulting Ag nanoparticles. The presence of pili led to the formation of smaller and more crystallized Ag nanoparticles. Overall, our findings underscore the intricate interplay of cytochromes, NADH, and pili in Ag(I) reduction. Such insights suggest potential strategies for further enhancing microbial Ag(I) reduction.

Significance of a minor pilin PilV in biofilm cohesion of Geobacter sulfurreducens.

Bacterial adhesion plays a vital role in forming and shaping the structure of electroactive biofilms that are essential for the performance of bioelectrochemical systems (BESs). Type IV pili are known to mediate cell adhesion in many Gram-negative bacteria, but the mechanism of pili-mediated cell adhesion of Geobacter species on anode surface remains unclear. Herein, a minor pilin PilV2 was found to be essential for cell adhesion ability of Geobacter sulfurreducens since the lack of pilV2 gene depressed the cell adhesion capability by 81.2% in microplate and the anodic biofilm density by 23.1 % at -0.1 V and 37.7 % at -0.3 V in BESs. The less cohesiveness of mutant biofilms increased the charge transfer resistance and biofilm resistance, which correspondingly lowered current generation of the pilV2-deficient strain by up to 63.2 % compared with that of the wild-type strain in BESs. The deletion of pilV2 posed an insignificant effect on the production of extracellular polysaccharides, pili, extracellular cytochromes and electron shuttles that are involved in biofilm formation or extracellular electron transfer (EET) process. This study demonstrated the significance of pilV2 gene in cell adhesion and biofilm formation of G. sulfurreducens, as well as the importance of pili-mediated adhesion for EET of electroactive biofilm.

Purification of Single-Stranded RNA Bacteriophages and Host Receptors for Structural Determination Using Cryo-Electron Microscopy.

Single-stranded RNA bacteriophages (ssRNA phages) are small viruses with a compact genome (~3-4 kb) that infect gram-negative bacteria via retractile pili. These phages have been applied in various fields since their discovery approximately 60 years ago. To understand their biology, it is crucial to analyze the structure of mature virions. Cryo-electron microscopy (cryo-EM) has been employed to determine the structures of two ssRNA phages, MS2 and Qß. This chapter presents a method for purifying these two phages and their receptor, the F-pilus, to allow examination using cryo-EM.

The ligand binding domain of a type IV pilus chemoreceptor PilJ has a different fold from that of another PilJ-type receptor McpN.

Bacterial chemoreceptors sense the extracellular signals and regulate bacterial motilities, biofilm formation, etc. The periplasmic ligand binding domains of chemoreceptors occur as different structural folds and recognize a diversity of chemical molecules. In Pseudomonas aeruginosa (PAO1), two bacterial chemoreceptors, McpN (PA2788) and PilJ (PA0411), are proposed to both contain a PilJ-like ligand-binding domain (LBD) (Pfam motif PF13675) and involved in nitrate chemotaxis and type IV pilus-mediated motility, respectively. The LBDs of McpN and PilJ consist of 135 and 263 residues, respectively, and share very low sequence identity, suggesting they might occur as different structures. Here, we found that PilJ-LBD folded into an HBM module, the same as the sensor domains of McpS-LBD and TorS-LBD, but it differed from that of McpN-LBD. We also observed a trimer in SEC and AUC and proposed a trimeric model based on the crystal structure. Based on the sequence, we classified the Pfam containing McpN-LBD and PilJ-LBD into three classes: sPilJ (single PilJ) represented by McpN-LBD with only one PilJ domain, dPilJ (dual PilJ) that contained dual PilJ domains, and hPilJ (hybrid PilJ) that comprises of a PilJ domain and another non-PilJ domain. Our work indicates a significant structural difference between the ligand binding domains of PilJ and McpN and will help our further study on both kinds of chemoreceptors.

Cryo-EM structures of type IV pili complexed with nanobodies reveal immune escape mechanisms.

Type IV pili (T4P) are prevalent, polymeric surface structures in pathogenic bacteria, making them ideal targets for effective vaccines. However, bacteria have evolved efficient strategies to evade type IV pili-directed antibody responses. Neisseria meningitidis are prototypical type IV pili-expressing Gram-negative bacteria responsible for life threatening sepsis and meningitis. This species has evolved several genetic strategies to modify the surface of its type IV pili, changing pilin subunit amino acid sequence, nature of glycosylation and phosphoforms, but how these modifications affect antibody binding at the structural level is still unknown. Here, to explore this question, we determine cryo-electron microscopy (cryo-EM) structures of pili of different sequence types with sufficiently high resolution to visualize posttranslational modifications. We then generate nanobodies directed against type IV pili which alter pilus function in vitro and in vivo. Cyro-EM in combination with molecular dynamics simulation of the nanobody-pilus complexes reveals how the different types of pili surface modifications alter nanobody binding. Our findings shed light on the impressive complementarity between the different strategies used by bacteria to avoid antibody binding. Importantly, we also show that structural information can be used to make informed modifications in nanobodies as countermeasures to these immune evasion mechanisms.

Subversion of a family of antimicrobial proteins by Salmonella enterica.

Salmonella enterica is a food-borne pathogen able to cause a wide spectrum of diseases ranging from mild gastroenteritis to systemic infections. During almost all stages of the infection process Salmonella is likely to be exposed to a wide variety of host-derived antimicrobial peptides (AMPs). AMPs are important components of the innate immune response which integrate within the bacterial membrane, thus forming pores which lead ultimately to bacterial killing. In contrast to other AMPs Bactericidal/Permeability-increasing Protein (BPI) displayed only weak bacteriostatic or bactericidal effects towards Salmonella enterica sv. Typhimurium (STM) cultures. Surprisingly, we found that sub-antimicrobial concentrations of BPI fold-containing (BPIF) superfamily members mediated adhesion of STM depending on pre-formed type 1 fimbriae. BPIF proteins directly bind to type 1 fimbriae through mannose-containing oligosaccharide modifications. Fimbriae decorated with BPIF proteins exhibit extended binding specificity, allowing for bacterial adhesion on a greater variety of abiotic and biotic surfaces likely promoting host colonization. Further, fimbriae significantly contributed to the resistance against BPI, probably through sequestration of the AMP before membrane interaction. In conclusion, functional subversion of innate immune proteins of the BPIF family through binding to fimbriae promotes Salmonella virulence by survival of host defense and promotion of host colonization.

Structural basis of Acinetobacter type IV pili targeting by an RNA virus.

Acinetobacters pose a significant threat to human health, especially those with weakened immune systems. Type IV pili of acinetobacters play crucial roles in virulence and antibiotic resistance. Single-stranded RNA bacteriophages target the bacterial retractile pili, including type IV. Our study delves into the interaction between Acinetobacter phage AP205 and type IV pili. Using cryo-electron microscopy, we solve structures of the AP205 virion with an asymmetric dimer of maturation proteins, the native Acinetobacter type IV pili bearing a distinct post-translational pilin cleavage, and the pili-bound AP205 showing its maturation proteins adapted to pilin modifications, allowing each phage to bind to one or two pili. Leveraging these results, we develop a 20-kilodalton AP205-derived protein scaffold targeting type IV pili in situ, with potential for research and diagnostics.

From isotopically labeled DNA to fluorescently labeled dynamic pili: building a mechanistic model of DNA transport to the cytoplasmic membrane.

SUMMARYNatural competence, the physiological state wherein bacteria produce proteins that mediate extracellular DNA transport into the cytosol and the subsequent recombination of DNA into the genome, is conserved across the bacterial domain. DNA must successfully translocate across formidable permeability barriers during import, including the cell membrane(s) and the cell wall, that are normally impermeable to large DNA polymers. This review will examine the mechanisms underlying DNA transport from the extracellular space to the cytoplasmic membrane. First, the challenges inherent to DNA movement through the cell periphery will be discussed to provide context for DNA transport during natural competence. The following sections will trace the development of a comprehensive model for DNA translocation to the cytoplasmic membrane, highlighting the crucial studies performed over the last century that have contributed to building contemporary DNA import models. Finally, this review will conclude by reflecting on what is still unknown about the process and the possible solutions to overcome these limitations.

The immune response of pregnant sow after vaccination with crude fimbriae (F4) extracts vaccine and immunoprotection of nursery pig against pathogenic E. coli (F4+ETEC).

BACKGROUND: Neonatal and post-weaning diarrhea is a concern disease caused by enterotoxigenic Escherichia coli fimbriae F4 (F4+ETEC) in pig farms. Diarrhea outbreaks are often severe and costly due to the high prevalence and spread of the disease within the same herd. Vaccine is one of strategic solution in protecting pig against F4+ETEC infection in particular pig farm. In present study, we conducted two trials of vaccination with crude F4 fimbriae extract vaccine in pregnant sow and nursery pigs. METHODS: In experiment 1 (20 sows; non-vaccinated control, n=10), we vaccinated pregnant sows (n=10) twice at 4 wk and 2 wk before farrowing and evaluated impact of vaccination on maternal immunity. The sow serum and colostrum were collected before vaccination, 2 and 4 weeks after vaccination, 6 hours after farrowing, respectively, and the piglet's serum from both groups (2 piglet/sow, 10 piglets from each group) were also collected on 3 days old to measure F4 specific IgG, F4 specific IgA using in house ELISA kit. In experiment 2, to optimize doses and dosage of candidate vaccine in piglets, 18 piglets (3 piglets/group) were allocated into five immunized groups and one control group (unimmunized group), we immunized piglets twice at 4 and 6 weeks old with difference doses (i.e., 0, 50, 100, 150, 200 µg), and for a dose 150 µg, we immunized with two dosages at 1 ml and 2 ml. Piglets were challenged with a 3 ml dose of 3 × 109 CFU/ml bacterial culture of enterotoxigenic Escherichia coli (F4+ETEC) in order to evaluate the efficacy of vaccine. After challenging, the clinical sign of the piglets was daily observed and the rectal swab was performed every day for investigation of the fecal shedding of Escherichia coli (F4+ETEC) by using PCR technique. Serum were collected before, 2 and 4 weeks after vaccination and 1 week after challenge to measure F4 specific IgG, F4 specific IgA using in house ELISA kit and cytokines levels (i.e., IL-1 beta, IL-6, IL-8 and TNF alpha) before and 1 week after challenge using commercial ELISA kit. RESULTS: The levels of antibody results showed that in experiment 1, the anti-F4 antibody levels both F4 specific IgG and F4 specific IgA in serum and colostrum of vaccinated sow increased significantly after vaccination. The piglets of immunized sows have antibody level both F4 specific IgG and F4 specific IgA in their serum higher than those piglets of unimmunized sows significantly (p < 0.01). In experiment 2, irrespective of different doses and dosage, there is no difference in term of F4 specific IgG and F4 specific IgA levels among immunized groups. However, all of vaccinated piglets showed F4 specific IgG and F4 specific IgA levels higher and the elimination of Escherichia coli (F4+ETEC) in feces post challenge faster (< 3 days) than unvaccinated group (> 5 days). For cytokines levels, a higher level of IL-1 beta, IL-6, IL-8 and TNF alpha at 1 week after challenge in vaccinated groups was found when compared with the levels in non-vaccinated group. CONCLUSIONS: Our results suggest that crude F4 fimbriae extract autogenous vaccine is a candidate vaccine for protecting piglets against diarrhea disease caused by enterotoxigenic Escherichia coli (F4+ETEC) and vaccination the pregnant sow twice before farrowing is one of strategies to provide maternal derived antibody to the newborn piglets for against enterotoxigenic Escherichia coli (F4+ETEC) during early life.