BACKGROUND
escherichia coli strains that cause diseases outside the intestine are known as extraintestinal pathogenic e. coli .
these strains include human uropathogenic e. coli , neonatal meningitis e. coli  and avian pathogenic e. coli  .
apec are frequently associated with extraintestinal infections in poultry, leading to respiratory or systemic diseases, which are responsible for large economic losses to the poultry industry worldwide  <cit> .
colibacillosis is a general term used to describe the large number of existing infections, including septicemia, cellulitis, omphalitis, peritonitis, respiratory tract infections, the egg yolk disease and swollen head syndrome.
colisepticemia, the most severe systemic disease, is characterized by pericarditis, perihepatitis and airsacculitis, and it leads to multiple organ failure and death  <cit> .
in apec, although several virulence factors such as adhesins, secretion and iron uptake systems, increased serum survival and cytotoxic proteins, have already been identified  <cit> , many others could exist and participate in the pathogenicity process.
these currently unknown virulence factors could play major roles in pathogenicity and could be significant for the development of measures for controlling the infectious processes.
adherence to and invasion of host cells are important steps in the pathogenesis of many bacteria  <cit> .
bacterial adherence is mediated by adhesins, which recognize receptors on the cell surface.
bacteria that invade host cells possess an important advantage in pathogenicity, overcoming the humoral immune response  <cit> .
a large family of bacterial outer membrane proteins facilitates the entry of the pathogen into host cells by allowing tight adherence to and invasion of the cells.
this family of proteins interacts with receptors displayed on the cell surface, triggering signaling cascades to rearrange the host cell cytoskeleton and induce the uptake of bacteria  <cit> .
the first two members of this family , although acting differently to promote the invasion of host cells, show significant sequence similarity, especially in the amino terminal region  <cit> .
the first invasin  to be described is produced by yersinia pseudotuberculosis and y. enterocolitica  <cit> , and it mediates bacterial entry into eukaryotic cells by high-affinity binding to members of the β <dig> integrin family  <cit> , which are heterodimeric integral membrane proteins that mediate communication between the extracellular environment and the cytoskeleton  <cit> .
the intimins, implicated in attaching and effacing lesions, are produced by enterohemorrhagic  and enteropathogenic e. coli   <cit> .
in contrast to invasin, the receptor for intimin binding is tir , a protein that is secreted into the host cell membrane by the bacterium itself  <cit> .
the intimins and invasins have similar domain structures: an n-terminal signal sequence, a conserved β-barrel domain, and a c-terminal passenger domain   <cit> .
the β-barrel structure is necessary for the passenger domain, which mediates interactions with host cells, to cross the outer membrane  <cit> .
the in silico analysis of recently sequenced genomes of some apec strains  enabled the identification of possible new virulence genes that could lead to a better understanding of the infectious process.
the in silico analysis of the sequenced genome of the apec strain sept <dig>  <cit>  identified a putative invasin gene, which is homologous to the not yet described ycho gene from e. coli str.
k- <dig> substr.
mg <dig>  and is present in  <dig> sequenced e. coli strains .
the role of this protein in pathogenicity or biological function of the apec strains was not previously established, and considering the importance of intimin/invasin-like proteins in other gram-negative pathogens, this work aimed to test the hypothesis that ycho might contribute to the pathogenesis of apec strain sept <dig>  in this work we showed that the ycho gene is highly expressed in the lungs and spleen during in vivo infection assays by strain sept <dig>  what suggests the importance of this gene in in vivo colonization of the host.
a mutant strain for gene ycho was constructed for in vivo and in vitro comparative analysis with the complemented strain and its counterpart wild-type strain.
in this study, we demonstrated, for the first time, that the gene ycho is expressed in vitro and in vivo and is involved in the bacterial capacity for adhesion to and invasion of cultivated cells in vitro, motility and biofilm formation and also influences the pathogenicity and the expression of many other genes.
these properties are important for pathogenicity in vivo, and our results suggest an important role for ycho in the pathogenesis of strain sept <dig>
RESULTS
in silico characterization of the putative invasin gene ycho
the apec strain sept <dig> was isolated from the liver of a laying hen presenting clinical signs of septicemia  <cit> .
a survey of the genome of strain sept <dig>  revealed a putative invasin homologous to the ycho gene from e. coli str.
k- <dig> substr.
mg <dig> .
to initiate this work, the re-annotation of this gene using rast  <cit>  was first performed, which showed a signal peptide sequence of 141 bp that was not present in the gi: <dig> protein annotated in the sept <dig> genome.
this peptide was considered to be part of the gene because it is known that this n-terminal signal sequence is an important domain of a typical invasin, predicted to mediate the translocation of the protein from the bacterial cytoplasm through the inner membrane  <cit> .
the ycho product alignment also showed that different e. coli pathotypes possess a peptide signal on this protein, reinforcing the decision to retain this sequence as part of the gene for the mutant construction.
this protein contains an n-terminal signal sequence, a conserved β-domain that forms a transmembrane β-barrel structure, and a c-terminal passenger domain that could be exported to the outside of the cell.
these domains together are the main characteristics of the intimin/invasin superfamily.
the intimin/invasin-like proteins normally form a longer structure, after the passenger domain, composed of repeated bacterial immunoglobulin-like domains .
in some cases, c-type lectin-like domains are present at the c-terminus  <cit> .
these structures were not found in the ycho protein.
ycho expression in vitro and in vivo
in this study, first, ycho expression was verified in the apec strain sept <dig> .
the results showed that this gene is expressed not only in culture conditions but is highly expressed in the lungs and spleen of chicks  <dig> and 48 h after inoculation, which suggests the importance of this gene in in vivo colonization of the host.
with these results, a mutant strain of the ycho gene and a complemented derivative strain containing the wild-type gene on a plasmid were constructed for comparative analysis with the wild-type apec strain sept <dig>  the mutant and complemented strains were verified by quantitative real time-pcr .
the high expression and high standard deviation of ycho expression in complemented strain is related to the number of plasmid copies present in this strain, which has an average of  <dig> copies per cell  <cit> .
a growth curve was constructed for the three strains, using lb and dmem media.
no differences were found between strains in both growing conditions .fig.
<dig> relative fold expression levels of ycho verified using quantitative real time-pcr .
expression of ycho in sept <dig> in the spleen and lungs after  <dig> and 48 h post infection compared to expression in vitro using lb media.
in vitro analysis of ycho expression in the sept <dig>  Δycho and complemented strains grown in dmem medium
the influence of the ycho gene on sept <dig> cell adherence
invasins are a class of proteins that allow bacteria to penetrate cells, normally by tight adherence to and invasion of eukaryotic cells  <cit> .
adherence and invasion are important steps for bacterial pathogenesis and contribute to the colonization, persistence and dissemination of a pathogen in the host organism  <cit> .
therefore, understanding the role of a possible invasin might help to elucidate the mechanisms of invasion and pathogenicity of host cells.
the capacity of the strains to adhere to chicken embryonic fibroblasts cell line cef was assessed to understand the possible influence of ycho gene on this phenotype.
because type  <dig> fimbriae binds to d-mannose residues and is important for apec adherence to chicken cells , adhesion assays were also performed in the presence of alpha-d-mannopyranoside , a potent non metabolized fimh antagonist.
the number of bacteria that adhered to pre-fixed cef cells was significantly lower for the ycho mutant than for the wild-type strain in the presence and absence of methyl-alpha-d-mannopyranoside .
the decrease in the presence of a d-mannose analog shows that the disorder was not due to a disturb in type  <dig> fimbriae expression.
the complemented strain restored the bacterial adhesion.
these data suggest that ycho is involved in sept <dig> adherence.
to investigate whether the change in adhesion ability was due to a disturbance in the expression of other genes possibly related to the adhesion capacity of this strain, different bacterial adhesin genes , that were identified by in silico analysis to exist in the genome of strain sept <dig>  were studied by qrt-pcr .
the expression levels of these genes were not significantly changed indicating that none of these genes are influenced by the lack of ycho, and the decreased adherence of the mutant strain is not due to a decreased expression of those genes.
thus, even without the bids and the c-type lectin-like domains  <cit> , the ycho probably acts as an adhesin in apec strain sept <dig> fig.
<dig> bacterial adhesion assay.
comparison of adhesion between the strains.
quantification of bacteria adhered to the chicken embryo fibroblast cell line  in the absence or presence of methyl-alpha-d-mannopyranoside.
relative fold expression of genes related to adhesion in sept <dig> verified using quantitative real time-pcr .
statistical significance was determined by tukey’s test in comparison with sept <dig>
gene ycho contributes to the invasion of chicken embryonic fibroblast cells
we assessed the ability of sept <dig> to invade the chicken embryonic fibroblast cell line cec- <dig> and the potential contribution of the ycho gene to this phenotype.
this cell line of avian origin was chosen because it resembles the environment bacteria would find in vivo.
sept <dig> was able to invade cec- <dig> cells efficiently, and the ycho gene played an important role in this phenotype.
the invasion assay was performed with two sets of cells: one set was lysed immediately after incubation with 50 μg ml− <dig> of gentamycin and the other was lysed after  <dig>  h of incubation with 5 μg ml− <dig> of gentamicyn.
the number of viable bacteria inside cells after the invasion assay was significantly lower in strain ∆ycho than in the wild-type strain, whereas the complemented strain restored this capacity .
the decreased invasiveness was significant, but was not completely abolished.
this results suggest that other genes reported to be related to invasion, such as flic , mota , bamb , ompa , ibeb , among others  that are present in this strain could also contribute to this process.
after  <dig>  hour of invasion, the number of surviving viable mutants was decreased while the wild-type and complemented strains retained their viability.
these data suggest that this protein plays a role not only in invasion, but also in bacterial survival in this cell type.fig.
<dig> invasion assay in cec- <dig>  comparison of strains after 1 h of incubation in 50 μg ml− <dig> gentamicin and survival after  <dig>  h in 5 μg ml− <dig> gentamicin.
statistical significance was determined by tukey’s test in comparison with the count of the strain sept <dig>
biofilm formation is influenced by ycho
biofilms are multicellular communities within a self-produced extracellular matrix attached to a surface  <cit> .
the extracellular matrix helps resist environmental changes and avoid host defenses.
given the capacity of strain sept <dig> to form biofilms on abiotic surfaces  <cit> , we assessed this ability in the mutant strain using the crystal violet biofilm test on polystyrene .
although the growth curves of strains ∆ycho and the c.∆ycho did not show any difference when compared with the wild-type strain , strain ∆ycho formed 50 % less biofilm than the wild-type strain after 24 h, indicating that ycho influences biofilm formation.
the complemented strain formed 35 % more biofilm than the wild-type strain, which is explained by the overexpression of the gene in its plasmidial structure and suggests the importance of this protein in biofilm formation.
these results corroborate a previous report in which a similar putative invasin protein of edwardsiella tarda was found to be essential for biofilm formation  <cit> , even though this protein contains a long structure of repeated bid, whereas ycho does not.fig.
<dig> biofilm formation.
cells were grown for 24 h in polystyrene plates with dmem.
averages followed by different letters are significantly different
the lack of ycho affects the virulence of strain sept362
in vivo assays were used to evaluate the pathogenicity of strain sept <dig> and its derivatives.
for this purpose, separated groups of one-day-old chicks  were infected with the mutant, its complemented derivative and the wild-type strain .
the percent survival of chicks infected with the wild-type was 70 % on the first day, 30 % on the third day, and 10 % at the end of the experiment.
for strain ∆ycho, the percent survival was 65 % on the first day, 60 % on the third day, and 40 % at the end of the seventh day.
no mortality was observed for the negative control infected with 109 cfu ml− <dig> of e. coli dh10β .
although there were no significant differences among the strains considering the seven days of the experiment , a significant change in the survival profile of the mutant strain compared to the wild-type was observed, after the second day .
the complemented strain presented a profile similar to that of the wild-type strain.
the lack of ycho gene decreases the virulence of strain sept <dig> after 48 h in the host, which indicates ycho has a role in survival of the strain in the host.
although this gene is also found in non pathogenic e. coli strains, all of these results suggest that the absence of gene ycho influences the virulence of the apec strain herein studied in a direct way.fig.
<dig> survival assay.
one-day-old broiler chicks were infected with 109 cfu ml− <dig> bacteria .
there was no statistical significance determined by the log-rank  test compared with the sept <dig> strain
it is known that apec pathogenesis is controlled by a number of virulence factors, including adhesins , iron acquisition systems , hemolysins and the temperature-sensitive hemagglutinin tsh, antibactericidal factors , and toxins  .
many of these virulence factors  have been found in strain sept <dig> by in silico search and in vivo analysis  <cit> , which most likely makes the pathogenicity of this strain multifactorial.
thus, ycho activity is one of several factors that could contribute to sept <dig> pathogenicity, and the full virulence observed in this strain would result from the sum of all the virulence factors present in it.
the lack of ycho affected the expression several genes in sept <dig> strain
the results presented so far showed that ycho gene has a pleiotropic effect on several biological characteristics, including those related to invasin like proteins, such as biofilm formation, adhesion to and invasion of in vitro cultured cells.
to further investigate the pleiotropic effect of gene ycho to all of these biological characteristics we used the transcriptome sequencing  analysis to compare the transcription profiles of the mutant strain and its wild-type strain.
we found that  <dig> genes were affected upon ycho deletion, which were classified into  <dig> broad categories  using ecocyc and uniprot.
most of affected genes are classified in metabolism , hypothetical  and membrane transport .
among the altered genes,  <dig>  % were downregulated in the mutant .
the rnaseq was validated by comparison of the expression levels of the genes csga, lpfa, fimh, flic, flhd, mota, flge, ecp and ycho by qrt-pcr technique.
the results were similar for both techniques .fig.
<dig> classification of the genes affected in ycho mutant, based on rnaseq analyses.
each gene is represented once, and is classified in the most relevant category.
percentage indicates the total number of affected factors in each category, relative to the total number of affected genes.
upregulated and downregulated genes classified in the most relevant category
the high expression of ycho in strain sept <dig> in lungs and spleens during infection and the possible membrane location of protein ycho suggest that the lack of this protein could generate a breakdown of the bacterial membrane, deregulating other membrane-located proteins and membrane transport proteins, triggering a pleiotropic effect, deregulating genes related to metabolism and thus several biological characteristics.
CONCLUSIONS
the adhesion, invasion and biofilm formation results presented in this paper together with the presence of the three basic structures of the protein , allow us to suggest that gene ycho protein plays roles as an adhesin/invasion in this specific strain.
despite its short passenger protein structure, without the bid, the protein is important for the virulence of apec strain sept <dig>  with the lack of ycho gene, there was a decrease in mortality in chicks.
these results, together with the alteration of the expression of  <dig> genes, led us to propose that ycho has a pleiotropic action in apec strain sept <dig> probably due to an imbalance effect on membrane structure what leads to the altered expression of membrane-related and metabolic genes.

 escherichia coli strains that cause diseases outside the intestine are known as extraintestinal pathogenic e. coli. these strains include human uropathogenic e. coli, neonatal meningitis e. coli and avian pathogenic e. coli. apec are frequently associated with extraintestinal infections in poultry, leading to respiratory or systemic diseases, which are responsible for large economic losses to the poultry industry worldwide. colibacillosis is a general term used to describe the large number of existing infections, including septicemia, cellulitis, omphalitis, peritonitis, respiratory tract infections, the egg yolk disease and swollen head syndrome. colisepticemia, the most severe systemic disease, is characterized by pericarditis, perihepatitis and airsacculitis, and it leads to multiple organ failure and death. in apec, although several virulence factors such as adhesins, secretion and iron uptake systems, increased serum survival and cytotoxic proteins, have already been identified, many others could exist and participate in the pathogenicity process. these currently unknown virulence factors could play major roles in pathogenicity and could be significant for the development of measures for controlling the infectious processes. adherence to and invasion of host cells are important steps in the pathogenesis of many bacteria. bacterial adherence is mediated by adhesins, which recognize receptors on the cell surface. bacteria that invade host cells possess an important advantage in pathogenicity, overcoming the humoral immune response. a large family of bacterial outer membrane proteins facilitates the entry of the pathogen into host cells by allowing tight adherence to and invasion of the cells. this family of proteins interacts with receptors displayed on the cell surface, triggering signaling cascades to rearrange the host cell cytoskeleton and induce the uptake of bacteria. the first two members of this family, although acting differently to promote the invasion of host cells, show significant sequence similarity, especially in the amino terminal region. the first invasin to be described is produced by yersinia pseudotuberculosis and y. enterocolitica, and it mediates bacterial entry into eukaryotic cells by high-affinity binding to members of the β integrin family, which are heterodimeric integral membrane proteins that mediate communication between the extracellular environment and the cytoskeleton. the intimins, implicated in attaching and effacing lesions, are produced by enterohemorrhagic and enteropathogenic e. coli. in contrast to invasin, the receptor for intimin binding is tir, a protein that is secreted into the host cell membrane by the bacterium itself. the intimins and invasins have similar domain structures: an n-terminal signal sequence, a conserved β-barrel domain, and a c-terminal passenger domain. the β-barrel structure is necessary for the passenger domain, which mediates interactions with host cells, to cross the outer membrane. the in silico analysis of recently sequenced genomes of some apec strains enabled the identification of possible new virulence genes that could lead to a better understanding of the infectious process. the in silico analysis of the sequenced genome of the apec strain sept identified a putative invasin gene, which is homologous to the not yet described ycho gene from e. coli str. k- substr. mg and is present in sequenced e. coli strains. the role of this protein in pathogenicity or biological function of the apec strains was not previously established, and considering the importance of intimin/invasin-like proteins in other gram-negative pathogens, this work aimed to test the hypothesis that ycho might contribute to the pathogenesis of apec strain sept in this work we showed that the ycho gene is highly expressed in the lungs and spleen during in vivo infection assays by strain sept what suggests the importance of this gene in in vivo colonization of the host. a mutant strain for gene ycho was constructed for in vivo and in vitro comparative analysis with the complemented strain and its counterpart wild-type strain. in this study, we demonstrated, for the first time, that the gene ycho is expressed in vitro and in vivo and is involved in the bacterial capacity for adhesion to and invasion of cultivated cells in vitro, motility and biofilm formation and also influences the pathogenicity and the expression of many other genes. these properties are important for pathogenicity in vivo, and our results suggest an important role for ycho in the pathogenesis of strain sept  in silico characterization of the putative invasin gene ycho the apec strain sept was isolated from the liver of a laying hen presenting clinical signs of septicemia. a survey of the genome of strain sept revealed a putative invasin homologous to the ycho gene from e. coli str. k- substr. mg. to initiate this work, the re-annotation of this gene using rast was first performed, which showed a signal peptide sequence of 141 bp that was not present in the gi: protein annotated in the sept genome. this peptide was considered to be part of the gene because it is known that this n-terminal signal sequence is an important domain of a typical invasin, predicted to mediate the translocation of the protein from the bacterial cytoplasm through the inner membrane. the ycho product alignment also showed that different e. coli pathotypes possess a peptide signal on this protein, reinforcing the decision to retain this sequence as part of the gene for the mutant construction. this protein contains an n-terminal signal sequence, a conserved β-domain that forms a transmembrane β-barrel structure, and a c-terminal passenger domain that could be exported to the outside of the cell. these domains together are the main characteristics of the intimin/invasin superfamily. the intimin/invasin-like proteins normally form a longer structure, after the passenger domain, composed of repeated bacterial immunoglobulin-like domains. in some cases, c-type lectin-like domains are present at the c-terminus. these structures were not found in the ycho protein. ycho expression in vitro and in vivo in this study, first, ycho expression was verified in the apec strain sept. the results showed that this gene is expressed not only in culture conditions but is highly expressed in the lungs and spleen of chicks and 48 h after inoculation, which suggests the importance of this gene in in vivo colonization of the host. with these results, a mutant strain of the ycho gene and a complemented derivative strain containing the wild-type gene on a plasmid were constructed for comparative analysis with the wild-type apec strain sept the mutant and complemented strains were verified by quantitative real time-pcr. the high expression and high standard deviation of ycho expression in complemented strain is related to the number of plasmid copies present in this strain, which has an average of copies per cell. a growth curve was constructed for the three strains, using lb and dmem media. no differences were found between strains in both growing conditions.fig. relative fold expression levels of ycho verified using quantitative real time-pcr. expression of ycho in sept in the spleen and lungs after and 48 h post infection compared to expression in vitro using lb media. in vitro analysis of ycho expression in the sept Δycho and complemented strains grown in dmem medium the influence of the ycho gene on sept cell adherence invasins are a class of proteins that allow bacteria to penetrate cells, normally by tight adherence to and invasion of eukaryotic cells. adherence and invasion are important steps for bacterial pathogenesis and contribute to the colonization, persistence and dissemination of a pathogen in the host organism. therefore, understanding the role of a possible invasin might help to elucidate the mechanisms of invasion and pathogenicity of host cells. the capacity of the strains to adhere to chicken embryonic fibroblasts cell line cef was assessed to understand the possible influence of ycho gene on this phenotype. because type fimbriae binds to d-mannose residues and is important for apec adherence to chicken cells, adhesion assays were also performed in the presence of alpha-d-mannopyranoside, a potent non metabolized fimh antagonist. the number of bacteria that adhered to pre-fixed cef cells was significantly lower for the ycho mutant than for the wild-type strain in the presence and absence of methyl-alpha-d-mannopyranoside. the decrease in the presence of a d-mannose analog shows that the disorder was not due to a disturb in type fimbriae expression. the complemented strain restored the bacterial adhesion. these data suggest that ycho is involved in sept adherence. to investigate whether the change in adhesion ability was due to a disturbance in the expression of other genes possibly related to the adhesion capacity of this strain, different bacterial adhesin genes, that were identified by in silico analysis to exist in the genome of strain sept were studied by qrt-pcr. the expression levels of these genes were not significantly changed indicating that none of these genes are influenced by the lack of ycho, and the decreased adherence of the mutant strain is not due to a decreased expression of those genes. thus, even without the bids and the c-type lectin-like domains, the ycho probably acts as an adhesin in apec strain sept fig. bacterial adhesion assay. comparison of adhesion between the strains. quantification of bacteria adhered to the chicken embryo fibroblast cell line in the absence or presence of methyl-alpha-d-mannopyranoside. relative fold expression of genes related to adhesion in sept verified using quantitative real time-pcr. statistical significance was determined by tukey’s test in comparison with sept gene ycho contributes to the invasion of chicken embryonic fibroblast cells we assessed the ability of sept to invade the chicken embryonic fibroblast cell line cec- and the potential contribution of the ycho gene to this phenotype. this cell line of avian origin was chosen because it resembles the environment bacteria would find in vivo. sept was able to invade cec- cells efficiently, and the ycho gene played an important role in this phenotype. the invasion assay was performed with two sets of cells: one set was lysed immediately after incubation with 50 μg ml− of gentamycin and the other was lysed after  h of incubation with 5 μg ml− of gentamicyn. the number of viable bacteria inside cells after the invasion assay was significantly lower in strain ∆ycho than in the wild-type strain, whereas the complemented strain restored this capacity. the decreased invasiveness was significant, but was not completely abolished. this results suggest that other genes reported to be related to invasion, such as flic, mota, bamb, ompa, ibeb, among others that are present in this strain could also contribute to this process. after  hour of invasion, the number of surviving viable mutants was decreased while the wild-type and complemented strains retained their viability. these data suggest that this protein plays a role not only in invasion, but also in bacterial survival in this cell type.fig. invasion assay in cec- comparison of strains after 1 h of incubation in 50 μg ml− gentamicin and survival after  h in 5 μg ml− gentamicin. statistical significance was determined by tukey’s test in comparison with the count of the strain sept biofilm formation is influenced by ycho biofilms are multicellular communities within a self-produced extracellular matrix attached to a surface. the extracellular matrix helps resist environmental changes and avoid host defenses. given the capacity of strain sept to form biofilms on abiotic surfaces, we assessed this ability in the mutant strain using the crystal violet biofilm test on polystyrene. although the growth curves of strains ∆ycho and the c.∆ycho did not show any difference when compared with the wild-type strain, strain ∆ycho formed 50 % less biofilm than the wild-type strain after 24 h, indicating that ycho influences biofilm formation. the complemented strain formed 35 % more biofilm than the wild-type strain, which is explained by the overexpression of the gene in its plasmidial structure and suggests the importance of this protein in biofilm formation. these results corroborate a previous report in which a similar putative invasin protein of edwardsiella tarda was found to be essential for biofilm formation, even though this protein contains a long structure of repeated bid, whereas ycho does not.fig. biofilm formation. cells were grown for 24 h in polystyrene plates with dmem. averages followed by different letters are significantly different the lack of ycho affects the virulence of strain sept362 in vivo assays were used to evaluate the pathogenicity of strain sept and its derivatives. for this purpose, separated groups of one-day-old chicks were infected with the mutant, its complemented derivative and the wild-type strain. the percent survival of chicks infected with the wild-type was 70 % on the first day, 30 % on the third day, and 10 % at the end of the experiment. for strain ∆ycho, the percent survival was 65 % on the first day, 60 % on the third day, and 40 % at the end of the seventh day. no mortality was observed for the negative control infected with 109 cfu ml− of e. coli dh10β. although there were no significant differences among the strains considering the seven days of the experiment, a significant change in the survival profile of the mutant strain compared to the wild-type was observed, after the second day. the complemented strain presented a profile similar to that of the wild-type strain. the lack of ycho gene decreases the virulence of strain sept after 48 h in the host, which indicates ycho has a role in survival of the strain in the host. although this gene is also found in non pathogenic e. coli strains, all of these results suggest that the absence of gene ycho influences the virulence of the apec strain herein studied in a direct way.fig. survival assay. one-day-old broiler chicks were infected with 109 cfu ml− bacteria. there was no statistical significance determined by the log-rank test compared with the sept strain it is known that apec pathogenesis is controlled by a number of virulence factors, including adhesins, iron acquisition systems, hemolysins and the temperature-sensitive hemagglutinin tsh, antibactericidal factors, and toxins. many of these virulence factors have been found in strain sept by in silico search and in vivo analysis, which most likely makes the pathogenicity of this strain multifactorial. thus, ycho activity is one of several factors that could contribute to sept pathogenicity, and the full virulence observed in this strain would result from the sum of all the virulence factors present in it. the lack of ycho affected the expression several genes in sept strain the results presented so far showed that ycho gene has a pleiotropic effect on several biological characteristics, including those related to invasin like proteins, such as biofilm formation, adhesion to and invasion of in vitro cultured cells. to further investigate the pleiotropic effect of gene ycho to all of these biological characteristics we used the transcriptome sequencing analysis to compare the transcription profiles of the mutant strain and its wild-type strain. we found that genes were affected upon ycho deletion, which were classified into broad categories using ecocyc and uniprot. most of affected genes are classified in metabolism, hypothetical and membrane transport. among the altered genes,  % were downregulated in the mutant. the rnaseq was validated by comparison of the expression levels of the genes csga, lpfa, fimh, flic, flhd, mota, flge, ecp and ycho by qrt-pcr technique. the results were similar for both techniques.fig. classification of the genes affected in ycho mutant, based on rnaseq analyses. each gene is represented once, and is classified in the most relevant category. percentage indicates the total number of affected factors in each category, relative to the total number of affected genes. upregulated and downregulated genes classified in the most relevant category the high expression of ycho in strain sept in lungs and spleens during infection and the possible membrane location of protein ycho suggest that the lack of this protein could generate a breakdown of the bacterial membrane, deregulating other membrane-located proteins and membrane transport proteins, triggering a pleiotropic effect, deregulating genes related to metabolism and thus several biological characteristics.  the adhesion, invasion and biofilm formation results presented in this paper together with the presence of the three basic structures of the protein, allow us to suggest that gene ycho protein plays roles as an adhesin/invasion in this specific strain. despite its short passenger protein structure, without the bid, the protein is important for the virulence of apec strain sept with the lack of ycho gene, there was a decrease in mortality in chicks. these results, together with the alteration of the expression of genes, led us to propose that ycho has a pleiotropic action in apec strain sept probably due to an imbalance effect on membrane structure what leads to the altered expression of membrane-related and metabolic genes. 
an in silico analysis of the genome of a septicemic apec strain demonstrated the presence of a putative invasin homologous to the ycho gene from e. coli str. mg in vitro and in vivo assays comparing a mutant strain carrying a null mutation of this gene, a complemented strain, and its counterpart wild-type strain showed that ycho plays a role in the pathogenicity of apec strain sept in vitro assays demonstrated that the mutant strain exhibited significant decreases in bacterial adhesiveness and invasiveness in chicken cells and biofilm formation. the results led us to propose that gene ycho contributes to the pathogenicity of apec strain sept influencing, in a pleiotropic manner, many biological characteristics, such as adhesion and invasion of in vitro cultured cells, biofilm formation and motility, which could be due to the possible membrane location of this protein. all of these results suggest that the absence of gene ycho would influence the virulence of the apec strain herein studied. invasins are members of a large family of outer membrane proteins that allow pathogen invasion into host cells by interacting with specific receptors on the cell surface. in vivo assay indicated a decrease in pathogenicity of the mutant strain. moreover, transcriptome analysis demonstrated that the ycho deletion affected the expression of genes.
 avian pathogenic escherichia coli strains cause extraintestinal diseases in birds, leading to substantial economic losses to the poultry industry worldwide. bacteria that invade cells can overcome the host humoral immune response, resulting in a higher pathogenicity potential. invasins are members of a large family of outer membrane proteins that allow pathogen invasion into host cells by interacting with specific receptors on the cell surface.  an in silico analysis of the genome of a septicemic apec strain demonstrated the presence of a putative invasin homologous to the ycho gene from e. coli str. k- substr. mg in vitro and in vivo assays comparing a mutant strain carrying a null mutation of this gene, a complemented strain, and its counterpart wild-type strain showed that ycho plays a role in the pathogenicity of apec strain sept in vitro assays demonstrated that the mutant strain exhibited significant decreases in bacterial adhesiveness and invasiveness in chicken cells and biofilm formation. in vivo assay indicated a decrease in pathogenicity of the mutant strain. moreover, transcriptome analysis demonstrated that the ycho deletion affected the expression of genes. among the altered genes,  % were downregulated in the mutant, including membrane proteins and metabolism genes.  the results led us to propose that gene ycho contributes to the pathogenicity of apec strain sept influencing, in a pleiotropic manner, many biological characteristics, such as adhesion and invasion of in vitro cultured cells, biofilm formation and motility, which could be due to the possible membrane location of this protein. all of these results suggest that the absence of gene ycho would influence the virulence of the apec strain herein studied. keywords escherichia coliapecychovirulencepathogenicityfapesp2012/04391-12012/09655-7dias da silveira wanderley issue-copyright-statement© the author 2016 