BACKGROUND
the tad  locus is present in many gram-positive and gram-negative bacteria as well as the archaea and likely represents an ancient subtype of type iv secretion systems that was horizontally transferred to many bacterial species early in the course of evolution.
the tad genes are located on a mobile genomic island coined "the widespread colonization island" by figurski and coworkers  <cit> .
the functions of the tad locus gene products have been best described for aggregatibacter  actinomycetemcomitans, where they are essential for adherence, biofilm formation, and pathogenesis.
the tad proteins are predicted to form a macromolecular transport system for the assembly and secretion of fimbria or pili, which mediate adherence of the organism to different surfaces  <cit> .
haemophilus ducreyi is a gram-negative coccobacillus that causes the sexually transmitted genital ulcer disease chancroid, which is a public health concern because it increases the risk of transmission and acquisition of human immunodeficiency virus- <dig>  h. ducreyi contains a  <dig>  kb flp  operon with  <dig> genes  that encode products with homology to proteins encoded by the tad locus in a. actinomycetemcomitans  <cit> .
h. ducreyi mutants that lack expression of either flp <dig> and flp <dig>  which encode fimbria like proteins, or tada, which has homology to ntpases of type iv secretion systems, have decreased abilities to attach to human foreskin fibroblasts  and to form microcolonies on hff  <cit> .
a mutant deficient in the expression of tada was significantly but partially attenuated in the temperature dependent rabbit model of experimental chancroid, while a mutant lacking flp <dig> and flp <dig> expression was fully virulent in this model  <cit> .
thus, whether flp-tad-mediated adherence and/or microcolony formation are critical factors in the virulence of h. ducreyi is unclear  <cit> .
in experimental and natural infection in humans, h. ducreyi forms aggregates, the first step in microcolony formation, and colocalizes with polymorphonuclear leukocytes and macrophages, which fail to ingest the organism.
in human inoculation experiments, a tada mutant is highly attenuated for virulence; whether the observed attenuation is due to the lack of secretion of the flp proteins or other unidentified effectors by the tad locus is unclear  <cit> .
given the discrepancy in virulence between the tada mutant and the flp1flp <dig> mutant in the temperature dependent rabbit model  <cit> , here we constructed and characterized a flp1- <dig> deletion mutant.
we tested the flp1- <dig> mutant for its ability to cause disease in human volunteers and its ability to form microcolonies and adhere to human fibroblasts.
our data indicate that expression of flps is required for virulence and that flp-tad mediated adherence correlates with the virulence of h. ducreyi in humans.
to our knowledge, this study is the first to provide definitive proof that expression of the flp proteins is required for the virulence of a bacterial pathogen in humans.
RESULTS
construction and characterization of 35000hpΔflp1-3
an unmarked, in frame deletion mutant of the flp <dig>  flp <dig>  and flp <dig> genes was constructed in 35000hp using recombineering technology and designated 35000hpΔflp1- <dig>  <cit> .
sequence analysis of 35000hpΔflp1- <dig> confirmed that flp <dig>  flp <dig> and flp <dig> had been replaced by a short orf that consisted of the upstream region of flp <dig>  the start codon of flp <dig>   <dig> bp encoding a scar peptide, and the last  <dig> bp of flp <dig>  including its stop codon.
by qrt-pcr, the expression levels of tada and tadg, two genes downstream of flp <dig>  were similar in 35000hpΔflp1- <dig> compared to 35000hp , suggesting that the remainder of the flp operon was normally transcribed.
35000hp and 35000hpΔflp1- <dig> demonstrated identical growth rates in broth .
the los profiles and omp patterns as analyzed by sds-page were similar for the mutant and the parent .
human inoculation experiments
to determine whether the flp proteins play a role in pathogenesis, 35000hpΔflp1- <dig> was compared with 35000hp for virulence using a mutant parent comparison trial in the human model of infection.
ten healthy adults  volunteered for the study.
three subjects  were inoculated in the first iteration, three subjects  in the second iteration, one subject  in the third iteration and three subjects  in the fourth iteration.
in the first iteration, each subject was infected with a fixed edd  of 35000hp at three sites on one arm and varying edds  of 35000hpΔflp1- <dig> on the other arm .
since the ability of the mutant to form pustules seemed impaired , we increased the dose of the mutant in subsequent iterations, as per protocol.
in the second iteration, volunteers were inoculated with  <dig> cfu of the parent and  <dig>   <dig>  and  <dig> cfu of the mutant .
in the third iteration, the volunteer was infected with  <dig> cfu of 35000hp and  <dig>   <dig>  and  <dig> cfu of the mutant .
in the fourth iteration, volunteers were inoculated with  <dig> cfu of the parent and  <dig>   <dig> and  <dig> cfu of the mutant .
initial
a, f = female, m = male; b, p = parent, m = mutant
the overall papule formation rate for the parent was 80%  at  <dig> sites and for the mutant was  <dig> %  at  <dig> sites .
mutant papules were significantly smaller  than were parent papules   <dig> h after inoculation .
the overall pustule formation rates were  <dig> %  at  <dig> parent sites and  <dig> %  at  <dig> mutant sites .
mutant pustules formed at only two sites in one volunteer.
these results indicate that expression of one or more of the flp <dig>  flp <dig>  and flp <dig> genes in the context of the intact secretion/assembly complex is necessary for h. ducreyi to initiate disease and progress to pustule formation in humans.
h. ducreyi was recovered intermittently from surface cultures.
of the  <dig> sites that were inoculated with the parent,  <dig>  yielded at least one positive surface culture, while  <dig> of  <dig> mutant sites  yielded a positive surface culture .
all colonies recovered from sites inoculated with the parent  or the mutant  and colonies from the parent  and mutant  inocula were tested for the presence of flp1-flp2-flp <dig> and fgba sequences by colony hybridization.
the fgba probe hybridized to all the colonies, while the flp1-2- <dig> probe hybridized only to the colonies obtained from the parent inoculated sites or the parent inocula.
thus, there was no cross contamination of mutant and parent sites during the course of the trial.
deletion of flp1flp2flp <dig> affects attachment of 35000hp to hff cells and microcolony formation
to confirm the phenotype of 35000hpΔflp1- <dig>  we complemented 35000hpΔflp1- <dig> in trans by electroporating it with plasmid pjw <dig>  which encodes the flp <dig>  flp <dig> and flp <dig>  flp <dig>  flp <dig>  and flp <dig> encode proteins with predicted molecular weights of  <dig>  kda,  <dig>  kda, and  <dig>  kda, respectively.
western blot analysis with a polyclonal sera that binds to flp <dig> and flp <dig> confirmed that 35000hpΔflp1- <dig> lacked the ability to express the flp <dig> and flp <dig> proteins  compared to 35000hp .
complementation of 35000hpΔflp1- <dig> with plasmid pjw <dig> resulted in restoration of the expression of the flp <dig> and flp <dig> proteins as determined by western blot .
35000hp, 35000hpΔflp1- <dig>  and 35000hpΔflp1- <dig> were also tested for their abilities to bind confluent hff monolayers.
35000hpΔflp1- <dig> significantly attached to hff cells at lower levels  than did 35000hp   .
35000hpΔflp1- <dig> adhered to hff cells  at significantly higher levels than 35000hpΔflp1- <dig>  and at similar levels as 35000hp  .
35000hp, 35000hpΔflp1- <dig>  and 35000hpΔflp1- <dig> were also compared for their abilities to form microcolonies after  <dig> h incubation with confluent hff monolayers.
35000hp formed numerous, densely populated microcolonies on the surfaces of hff cells  <cit>  .
35000hpΔflp1- <dig> formed sparse and very small microcolonies  when compared to 35000hp; the complemented mutant demonstrated a restored phenotype similar to 35000hp .
thus, complementation of the mutant restored the parental phenotypes.
discussion
for this study, we focused on whether the expression of the flp proteins was necessary for virulence of h. ducreyi.
we constructed an unmarked, in frame deletion mutant lacking the flp1flp2flp <dig> genes in 35000hp using a recombineering strategy  <cit>  and found that 35000hpΔflp1- <dig> was significantly impaired in its ability to cause disease in the human model of infection.
flp1- <dig> joins hgba, dsra, ncaa, lspa1-lspa <dig>  pal, tada sapbc and cpxa as the ninth gene required for full virulence by h. ducreyi in the human inoculation experiments  <cit>  .
the flp-tad gene cluster is constitutively transcribed as a single polycistronic operon in vitro  <cit> .
relative to its expression during in vitro growth, tada transcripts are enriched in experimental pustules, suggesting that the flp-tad operon is upregulated in vivo  <cit> .
cpxra is the only obvious intact two-component signal transduction system contained in h. ducreyi.
transcription of flp1- <dig> and several other major virulence determinants are negatively regulated by conditions that favor phosphorylation of cpxr  <cit> .
purified recombinant cpxr interacts with the promoter regions of the flp operon in electrophoretic mobility shift assays  <cit> .
deletion of cpxa leads to loss of cpxa phosphatase activity, activates cpxr, and cripples the ability of h. ducreyi to infect humans  <cit> .
in contrast, a cpxr deletion mutant has no effect on or upregulates the expression of virulence determinants and is fully virulent in human volunteers  <cit> .
taken together, the data suggest that the flp-tad operon may be upregulated in vivo due to downregulation of cpxra.
the human inoculation experiments are limited in that we are precluded by several regulatory bodies from testing trans-complemented mutants in humans.
however, complementation of 35000hpΔflp1- <dig> in trans restored the ability of the mutant to form microcolonies and bind to hff cells, suggesting that the phenotype of the mutant is due to the deletion of the flp genes.
in the human inoculation experiments, we use 35000hp to examine the role of virulence factors in h. ducreyi pathogenesis.
there are two classes of h. ducreyi strains, which express different immunotypes and proteomes  <cit> .
although we were able to amplify flp1- <dig> alleles from six class i and three class ii strains , attempts to sequence the amplicons were unsuccessful, so we do not know if there is a difference in the flp genes in the class i and class ii strains.
35000hp is a class i strain; whether the flp proteins play a role in the virulence of class ii strains is unknown.
we previously reported that a tada mutant is attenuated for pustule formation in the human challenge model  <cit> .
however, the tada mutant, but not a flp1flp <dig> double mutant, is attenuated in the rabbit model of chancroid  <cit> .
nika et al previously reported that both the flp1flp <dig> mutant and the tada mutant demonstrate decreased abilities to attach to hff cells and form fewer microcolonies on hff cells  <cit> .
these data suggested that microcolony formation by itself is not a virulence factor for h. ducreyi.
although h. ducreyi does not appear to co-localize with fibroblasts in experimental or natural chancroid  <cit> , our data indicate that adherence to hff cells in vitro correlates with the virulence of h. ducreyi in humans.
similarly, both flp <dig> and tada mutants fail to colonize or cause disease in a rat infection model with a. actinomycetemcomitans  <cit> , and both flp <dig> mutant and tadd mutants of pasteurella multocida are attenuated in the septicemic mouse model  <cit> .
in a. actinomycetemcomitans, flp pili are assembled as bundles of long fibers in which flp <dig> is the major structural component  <cit> .
however, there is no evidence that the flp proteins are assembled into a pilus-like structure in h. ducreyi  <cit> .
several bacterial species including a. actinomycetemcomitans have two flp genes  <cit> .
h. ducreyi contains three flp genes, which have between 50-80% similarity to one another  <cit> .
deletion of flp <dig> and flp <dig> results in decreased adherence of h. ducreyi to hff cells and subsequent microcolony formation  <cit> ; the function of flp <dig> is unclear.
in vitro, h. ducreyi forms microcolonies, a key step in biofilm formation.
in vivo, h. ducreyi forms aggregates and colocalizes with macrophages, pmns, collagen and fibrin  <cit> .
h. ducreyi contains a luxs homologue that has autoinducer  activity in a vibrio harveyi-based reporter system, and a luxs mutant is partially attenuated for virulence in human volunteers  <cit> .
taken together, these data suggest that the formation of microcolonies, aggregates and quorum sensing mechanisms may be important for h. ducreyi pathogenesis.
whether the flp proteins contribute to this process by mediating attachment to host cells or initiating microcolony formation in the skin remains a subject for future investigation.
CONCLUSIONS
we have constructed an unmarked, in frame deletion mutant lacking the flp1flp2flp <dig> genes in h. ducreyi strain 35000hp.
the deletion mutant, 35000hpΔflp1- <dig>  has an intact tad secretion system.
our data show that production and secretion of the flp proteins contributes to microcolony formation and attachment of 35000hp to hff cells in vitro.
complementation of the mutant with flp1- <dig> in trans restored the parental phenotype.
additionally, expression of flp1- <dig> is necessary for h. ducreyi to initiate disease and progress to pustule formation in humans.
future studies will focus on how flp proteins contribute to microcolony formation and attachment in vivo.

 the tad locus is present in many gram-positive and gram-negative bacteria as well as the archaea and likely represents an ancient subtype of type iv secretion systems that was horizontally transferred to many bacterial species early in the course of evolution. the tad genes are located on a mobile genomic island coined "the widespread colonization island" by figurski and coworkers. the functions of the tad locus gene products have been best described for aggregatibacter actinomycetemcomitans, where they are essential for adherence, biofilm formation, and pathogenesis. the tad proteins are predicted to form a macromolecular transport system for the assembly and secretion of fimbria or pili, which mediate adherence of the organism to different surfaces. haemophilus ducreyi is a gram-negative coccobacillus that causes the sexually transmitted genital ulcer disease chancroid, which is a public health concern because it increases the risk of transmission and acquisition of human immunodeficiency virus- h. ducreyi contains a kb flp operon with genes that encode products with homology to proteins encoded by the tad locus in a. actinomycetemcomitans. h. ducreyi mutants that lack expression of either flp and flp which encode fimbria like proteins, or tada, which has homology to ntpases of type iv secretion systems, have decreased abilities to attach to human foreskin fibroblasts and to form microcolonies on hff. a mutant deficient in the expression of tada was significantly but partially attenuated in the temperature dependent rabbit model of experimental chancroid, while a mutant lacking flp and flp expression was fully virulent in this model. thus, whether flp-tad-mediated adherence and/or microcolony formation are critical factors in the virulence of h. ducreyi is unclear. in experimental and natural infection in humans, h. ducreyi forms aggregates, the first step in microcolony formation, and colocalizes with polymorphonuclear leukocytes and macrophages, which fail to ingest the organism. in human inoculation experiments, a tada mutant is highly attenuated for virulence; whether the observed attenuation is due to the lack of secretion of the flp proteins or other unidentified effectors by the tad locus is unclear. given the discrepancy in virulence between the tada mutant and the flp1flp mutant in the temperature dependent rabbit model, here we constructed and characterized a flp1- deletion mutant. we tested the flp1- mutant for its ability to cause disease in human volunteers and its ability to form microcolonies and adhere to human fibroblasts. our data indicate that expression of flps is required for virulence and that flp-tad mediated adherence correlates with the virulence of h. ducreyi in humans. to our knowledge, this study is the first to provide definitive proof that expression of the flp proteins is required for the virulence of a bacterial pathogen in humans.  construction and characterization of 35000hpΔflp1-3 an unmarked, in frame deletion mutant of the flp flp and flp genes was constructed in 35000hp using recombineering technology and designated 35000hpΔflp1-. sequence analysis of 35000hpΔflp1- confirmed that flp flp and flp had been replaced by a short orf that consisted of the upstream region of flp the start codon of flp bp encoding a scar peptide, and the last bp of flp including its stop codon. by qrt-pcr, the expression levels of tada and tadg, two genes downstream of flp were similar in 35000hpΔflp1- compared to 35000hp, suggesting that the remainder of the flp operon was normally transcribed. 35000hp and 35000hpΔflp1- demonstrated identical growth rates in broth. the los profiles and omp patterns as analyzed by sds-page were similar for the mutant and the parent. human inoculation experiments to determine whether the flp proteins play a role in pathogenesis, 35000hpΔflp1- was compared with 35000hp for virulence using a mutant parent comparison trial in the human model of infection. ten healthy adults volunteered for the study. three subjects were inoculated in the first iteration, three subjects in the second iteration, one subject in the third iteration and three subjects in the fourth iteration. in the first iteration, each subject was infected with a fixed edd of 35000hp at three sites on one arm and varying edds of 35000hpΔflp1- on the other arm. since the ability of the mutant to form pustules seemed impaired, we increased the dose of the mutant in subsequent iterations, as per protocol. in the second iteration, volunteers were inoculated with cfu of the parent and and cfu of the mutant. in the third iteration, the volunteer was infected with cfu of 35000hp and and cfu of the mutant. in the fourth iteration, volunteers were inoculated with cfu of the parent and and cfu of the mutant. initial a, f = female, m = male; b, p = parent, m = mutant the overall papule formation rate for the parent was 80% at sites and for the mutant was % at sites. mutant papules were significantly smaller than were parent papules h after inoculation. the overall pustule formation rates were % at parent sites and % at mutant sites. mutant pustules formed at only two sites in one volunteer. these results indicate that expression of one or more of the flp flp and flp genes in the context of the intact secretion/assembly complex is necessary for h. ducreyi to initiate disease and progress to pustule formation in humans. h. ducreyi was recovered intermittently from surface cultures. of the sites that were inoculated with the parent, yielded at least one positive surface culture, while of mutant sites yielded a positive surface culture. all colonies recovered from sites inoculated with the parent or the mutant and colonies from the parent and mutant inocula were tested for the presence of flp1-flp2-flp and fgba sequences by colony hybridization. the fgba probe hybridized to all the colonies, while the flp1-2- probe hybridized only to the colonies obtained from the parent inoculated sites or the parent inocula. thus, there was no cross contamination of mutant and parent sites during the course of the trial. deletion of flp1flp2flp affects attachment of 35000hp to hff cells and microcolony formation to confirm the phenotype of 35000hpΔflp1- we complemented 35000hpΔflp1- in trans by electroporating it with plasmid pjw which encodes the flp flp and flp flp flp and flp encode proteins with predicted molecular weights of kda, kda, and kda, respectively. western blot analysis with a polyclonal sera that binds to flp and flp confirmed that 35000hpΔflp1- lacked the ability to express the flp and flp proteins compared to 35000hp. complementation of 35000hpΔflp1- with plasmid pjw resulted in restoration of the expression of the flp and flp proteins as determined by western blot. 35000hp, 35000hpΔflp1- and 35000hpΔflp1- were also tested for their abilities to bind confluent hff monolayers. 35000hpΔflp1- significantly attached to hff cells at lower levels than did 35000hp. 35000hpΔflp1- adhered to hff cells at significantly higher levels than 35000hpΔflp1- and at similar levels as 35000hp. 35000hp, 35000hpΔflp1- and 35000hpΔflp1- were also compared for their abilities to form microcolonies after h incubation with confluent hff monolayers. 35000hp formed numerous, densely populated microcolonies on the surfaces of hff cells. 35000hpΔflp1- formed sparse and very small microcolonies when compared to 35000hp; the complemented mutant demonstrated a restored phenotype similar to 35000hp. thus, complementation of the mutant restored the parental phenotypes. discussion for this study, we focused on whether the expression of the flp proteins was necessary for virulence of h. ducreyi. we constructed an unmarked, in frame deletion mutant lacking the flp1flp2flp genes in 35000hp using a recombineering strategy and found that 35000hpΔflp1- was significantly impaired in its ability to cause disease in the human model of infection. flp1- joins hgba, dsra, ncaa, lspa1-lspa pal, tada sapbc and cpxa as the ninth gene required for full virulence by h. ducreyi in the human inoculation experiments. the flp-tad gene cluster is constitutively transcribed as a single polycistronic operon in vitro. relative to its expression during in vitro growth, tada transcripts are enriched in experimental pustules, suggesting that the flp-tad operon is upregulated in vivo. cpxra is the only obvious intact two-component signal transduction system contained in h. ducreyi. transcription of flp1- and several other major virulence determinants are negatively regulated by conditions that favor phosphorylation of cpxr. purified recombinant cpxr interacts with the promoter regions of the flp operon in electrophoretic mobility shift assays. deletion of cpxa leads to loss of cpxa phosphatase activity, activates cpxr, and cripples the ability of h. ducreyi to infect humans. in contrast, a cpxr deletion mutant has no effect on or upregulates the expression of virulence determinants and is fully virulent in human volunteers. taken together, the data suggest that the flp-tad operon may be upregulated in vivo due to downregulation of cpxra. the human inoculation experiments are limited in that we are precluded by several regulatory bodies from testing trans-complemented mutants in humans. however, complementation of 35000hpΔflp1- in trans restored the ability of the mutant to form microcolonies and bind to hff cells, suggesting that the phenotype of the mutant is due to the deletion of the flp genes. in the human inoculation experiments, we use 35000hp to examine the role of virulence factors in h. ducreyi pathogenesis. there are two classes of h. ducreyi strains, which express different immunotypes and proteomes. although we were able to amplify flp1- alleles from six class i and three class ii strains, attempts to sequence the amplicons were unsuccessful, so we do not know if there is a difference in the flp genes in the class i and class ii strains. 35000hp is a class i strain; whether the flp proteins play a role in the virulence of class ii strains is unknown. we previously reported that a tada mutant is attenuated for pustule formation in the human challenge model. however, the tada mutant, but not a flp1flp double mutant, is attenuated in the rabbit model of chancroid. nika et al previously reported that both the flp1flp mutant and the tada mutant demonstrate decreased abilities to attach to hff cells and form fewer microcolonies on hff cells. these data suggested that microcolony formation by itself is not a virulence factor for h. ducreyi. although h. ducreyi does not appear to co-localize with fibroblasts in experimental or natural chancroid, our data indicate that adherence to hff cells in vitro correlates with the virulence of h. ducreyi in humans. similarly, both flp and tada mutants fail to colonize or cause disease in a rat infection model with a. actinomycetemcomitans, and both flp mutant and tadd mutants of pasteurella multocida are attenuated in the septicemic mouse model. in a. actinomycetemcomitans, flp pili are assembled as bundles of long fibers in which flp is the major structural component. however, there is no evidence that the flp proteins are assembled into a pilus-like structure in h. ducreyi. several bacterial species including a. actinomycetemcomitans have two flp genes. h. ducreyi contains three flp genes, which have between 50-80% similarity to one another. deletion of flp and flp results in decreased adherence of h. ducreyi to hff cells and subsequent microcolony formation; the function of flp is unclear. in vitro, h. ducreyi forms microcolonies, a key step in biofilm formation. in vivo, h. ducreyi forms aggregates and colocalizes with macrophages, pmns, collagen and fibrin. h. ducreyi contains a luxs homologue that has autoinducer activity in a vibrio harveyi-based reporter system, and a luxs mutant is partially attenuated for virulence in human volunteers. taken together, these data suggest that the formation of microcolonies, aggregates and quorum sensing mechanisms may be important for h. ducreyi pathogenesis. whether the flp proteins contribute to this process by mediating attachment to host cells or initiating microcolony formation in the skin remains a subject for future investigation.  we have constructed an unmarked, in frame deletion mutant lacking the flp1flp2flp genes in h. ducreyi strain 35000hp. the deletion mutant, 35000hpΔflp1- has an intact tad secretion system. our data show that production and secretion of the flp proteins contributes to microcolony formation and attachment of 35000hp to hff cells in vitro. complementation of the mutant with flp1- in trans restored the parental phenotype. additionally, expression of flp1- is necessary for h. ducreyi to initiate disease and progress to pustule formation in humans. future studies will focus on how flp proteins contribute to microcolony formation and attachment in vivo. 
these data suggest that production and secretion of the flp proteins contributes to microcolony formation and attachment to hff cells in vitro. future studies will focus on how flp proteins contribute to microcolony formation and attachment in vivo. h. ducreyi mutants that lack expression of flp and flp or tada, which has homology to ntpases of type iv secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts. the overall papule formation rate for the parent was 80% and for the mutant was %. a tada mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp mutant is virulent in rabbits. we constructed 35000hpΔflp1- a deletion mutant that lacks expression of all three flp proteins but has an intact tad secretion system. the overall pustule formation rates were % at parent sites and % at mutant sites. expression of flp1- is also necessary for h. ducreyi to initiate disease and progress to pustule formation in humans. complementation of the mutant with flp1- in trans restored the parental phenotype. mutant papules were significantly smaller than were parent papules h after inoculation. haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp operon that encodes proteins predicted to contribute to adherence and pathogenesis. whether a flp deletion mutant would cause disease in humans is not clear.
 haemophilus ducreyi, the causative agent of the sexually transmitted disease chancroid, contains a flp operon that encodes proteins predicted to contribute to adherence and pathogenesis. h. ducreyi mutants that lack expression of flp and flp or tada, which has homology to ntpases of type iv secretion systems, have decreased abilities to attach to and form microcolonies on human foreskin fibroblasts. a tada mutant is attenuated in its ability to cause disease in human volunteers and in the temperature dependent rabbit model, but a flp1flp mutant is virulent in rabbits. whether a flp deletion mutant would cause disease in humans is not clear.  we constructed 35000hpΔflp1- a deletion mutant that lacks expression of all three flp proteins but has an intact tad secretion system. 35000hpΔflp1- was impaired in its ability to form microcolonies and to attach to hff in vitro when compared to its parent. complementation of the mutant with flp1- in trans restored the parental phenotype. to test whether expression of flp1- was necessary for virulence in humans, ten healthy adult volunteers were experimentally infected with a fixed dose of 35000hp on one arm and three doses of 35000hpΔflp1- on the other arm. the overall papule formation rate for the parent was 80% and for the mutant was %. mutant papules were significantly smaller than were parent papules h after inoculation. the overall pustule formation rates were % at parent sites and % at mutant sites.  these data suggest that production and secretion of the flp proteins contributes to microcolony formation and attachment to hff cells in vitro. expression of flp1- is also necessary for h. ducreyi to initiate disease and progress to pustule formation in humans. future studies will focus on how flp proteins contribute to microcolony formation and attachment in vivo. 