Abstract Pig Actinobacillus pleuropneumoniae （App） could induce chronic respiratory tract infection in pigs， which causes major economic losses on pig industry. Bacterial biofilm （BBF） is bacterial community adsorbed on the surface of biomaterials or body cavity， to protect bacteria escape， and recurrent outbreaks of related infectious diseases and chronic infections resulting therefrom are called bacterial biofilm diseases. App BF belongs to polymers with spatial structure in vitro， and its formation is regulated by multiple genes. Among them， gene deletion of the key component TolC of multidrug efflux pumps and type I secretion systems causes that App BF adhesion weakens; gene deletion of catalytic core ClpP of Clp proteolytic complex induces the inhibition of BF formation; outer membrane lipoprotein VacJ of App promotes BF formation; gene deletion of active enzyme LuxS enhances the formation of App BF and decreases bacterial adhesion ability; gene deletion of Adh obviously declines bacterial accumulation， BF formation and adhesion to host cells. In this paper， BF formation or inhibition mechanism in App is elaborated from molecular level， which could provide reference basis for exploring the prevention of its biofilm diseases.
　　Key words Pig; A. pleuropneumoniae; Biofilm （BF）; Gene regulation; Formation mechanism; Inhibition
　　Actinobacillus pleuropneumoniae （App） is pathogen of porcine contagious pleuropneumonia， and belongs to the member of Pasteurellaceae family. App spreads through aerosol or direct contact in infected animals， and could induce dyspnea and even rapid death， which causes great economic losses on pig industry[1]. Animals exposed to App may develop chronic infections or become asymptomatic carriers， becoming the source of disease transmission. App virulence factors contain type ？ flagellum， polysaccharides， poreforming toxins， Apx toxin， etc. Polysaccharides also could be divided into lipopolysaccharide （LPS）， capsular polysaccharide （CPS） and polyNacetyl glucosamine polymer （PGA）， and they all exist in biofilm （BF） matrix. Pathogenic effect of BF in App is gradually accepted[2-3].
　　Under specific condition， BF is regulated by genes of App and external environment. App forms bacterial community on biological surfaces （e.g. mucosa layer） and nonbiological surfaces （e.g. farm floor or equipment）， and it is surrounded by selfproduced polysaccharides， proteins and nucleic acids. Therefore， BF protects bacteria from harsh environments. Bacteria in BF could resist immune response attacks of host， and are not sensitive to dehydration and bactericides. Repeated outbreaks of related infectious diseases and chronic infections resulting therefrom are called BF diseases， which is extremely difficult to be treated and prevented in clinic. Researches also show that the isolated App BF strain has stronger drug resistance to ampicillin， florfenicol， tamicin and telmicocin than non BF strain. Related explanations on drug resistance mechanism of BF are as below. First， nutrition restriction. The slowing of strain growth velocity， consumption of nutrients and oxygen， and accumulation of metabolic wastes in BF all could promote bacteria in a kind of nongrowth status， also known as starvation status， and bacteria in the status are almost completely insensitive to antibiotics that inhibit their growth. Second， antibiotic osmosis barrier theory. Obvious characteristics of BF are that bacterial density is high， and the space between bacteria is narrow， and it can also synthesize extracellular matrix which differs greatly from single bacteria in quantity and composition. Waterinsoluble extracellular polysaccharides are the main components， which constitutes external environment for the growth of biofilm bacteria， and its 3D structure could effectively protect biofilm bacteria. A large number of extracellular matrix and narrow space between strains in BF become a barrier that prevents antibiotics from penetrating BF[4]. Based on prior literatures， BF formation and inhibition mechanism of App are summarized， which aims to provide reference basis for better treating or preventing the disease. 　　BF maturation
　　Extracellular polymers （EPS） are consisted of extracellular polysaccharides， extracellular DNA and extracellular protein， which could occupy more than 90% of BF dry weight. The secretion of EPS plays a key role in bacteria and BF formation. The compositions of extracellular polysaccharides vary greatly， making skeleton structure of bacterial BF play an important role in interbacterial adhesion， maturation and diffusion. Extracellular DNA is structural component of BF matrix in some bacteria， and plays a key role in BF formation and maintenance. Extracellular proteins of BF matrix （containing extracellular enzyme， structural protein and protein appendages） often join in integrity and stability of polysaccharide matrix network[5]. It is clear that EPS constitutes the bracket for BF threedimensional structure， and plays an important role in surface adhesion and condensation.
　　BF formation is related to bacteria species and different living conditions. Generally， BF formation starts from cell surface adhesion and the generation of EPS， which further attracts other bacteria. After massive proliferation， colony is generated， and it finally develops a mature BF. At early stage， various extracellular viscous substances such as extracellular DNA， adhesin and bacterial surface structure （e.g. flagellum and pili） start adhesion. The hydrophobicity of bacterial cell surface affects the colony aggregation， and the secretion of extracellular matrix further enhances the adhesion. By switching to gene expression model， bacteria generate EPS and intercellular signaling molecules， thereby causing the maturation of BF in complex 3D structure[4-5].
　　TolC gene regulation
　　TolC is the key component of multidrug efflux pumps and type I secretion systems， and plays a key role in multidrug resistance and virulence of many gramnegative bacteria[6]. It is found that TolC joins in BF formation of Salmonella typhimurium and Escherichia coli[7]. The dynamics of BF formation by different App isolated strains is different. By comparing App wild type （WT） and TolC gene deletion type （┐TolC）， it is found that ┐TolC shows initial adhesion defect. The first step of BF formation is bacteria adhering and aggregating to biological surfaces， and hydrophobicity and movement of cell surface play an important role in bacterial adhesion[4]. Compared with WT， cell hydrophobicity in ┐TolC declines， further affecting adherence of bacteria.
　　Compared with WT， BF components of ┐TolC always lack extracellular proteins in whole formation process of BF， and a potential mechanism of the phenotype is inactive TolC gene inducing the decrease of secretory protein. In the initial stage of BF formation， ┐TolC generates little PGA， and it may help to reduce surface adhesion. As efflux pump and type I secretion system， TolC has been studied in detail， and many substances such as antibiotics， toxins and signaling molecules are exhausted via it[8]. Research proves that TolC system in wild type may secrete some unknown soluble BF promoters. Via crossfeed in ┐TolC， BF formation is recovered partially. BF promoter may be a densitysensing molecule， which affects initial adhesion and maturation of BF. It has been proved that TolC is crucial to the secretion of ApxII （ hemolysin）， while  hemolysin is essential for BF formation of Staphylococcus aureus and Streptococcus pneumoniae[6， 9]. It needs further studying if ApxII uses TolC to save BF formation in ┐TolC. It is found that TolC has an important role in the formation of App BF， and the deletion of TolC could cause initial adhesion of App weakening. It is speculated that there are several factors promoting adhesion ability of BF to weaken in initial stage of ┐TolC ， and one of them is PGA decrease making that cell surface hydrophobicity in ┐TolC declines， and the decrease of extracellular proteins in BF matrix also affects the formation of BF. TolC affects initial adhesion of BF cell， thereby affecting the formation of BF in App. These findings provide important thinking for studying that TolC resists BF in App[6]. 　　ClpP gene regulation
　　In the respiratory tract and lung tissue of pigs， App needs maintaining a physiological balance reaction to respond to various challenges. ClpP is also called catalytic core of Clp proteolytic complex， and joins in various stress responses and regulation of BF formation in many pathogens. To explore the effect of ClpP in App virulence， ClpP gene was deleted by homologous recombination， and mutant strain S8┐ClpP was generated. Under high temperature and stress condition， proliferation rate of mutant strain S8┐ClpP slowed down. It showed that ClpP protein was necessary for App stress tolerance. Interestingly， compared with WT， mutant strain S8┐ClpP in vitro displayed that the ability of absorbing iron ions enhanced. Via scanning electron microscope， it was observed that bacteria surface was rough and irregular， and cell volume increased. Confocal laser scanning microscope displayed that the formation of BF in mutant strain S8┐ClpP was inhibited. Via RNA sequencing， transcriptional spectra of WT S8 and S8┐ClpP were studied. The results showed that there were 16 genes changing expression due to ClpP gene deletion. These data display that ClpP has an important role in stress response， iron ion uptake， cell morphology and BF formation， and it may also play a role in regulating the virulence factors of App[10].
　　VacJ gene regulation
　　Epimembrane protein of App mediates infection， and is action site of defense system of host. Outer membrane lipoprotein （VacJ） of App plays a role in resisting serum and intercellular transmission. To explore the effect of VacJ in App pathopoiesis， VacJ gene deletion mutant md12┐VacJ was constructed. Susceptibility of the mutant to potassium chloride， sodium dodecyl sulfonate and several antibiotics increased， which indicated that VacJ gene mutation impaired the stability of outer membrane. Nonprotein nitrogen fluorescence increase and significant cell morphological variation of mutant md12┐VacJ further verified that VacJ played a key role in maintaining integrity of App outer membrane. Additionally， compared with WT， the resistance of mutant md12┐VacJ to serum and complement declined. Interestingly， BF formation of mutant md12┐VacJ was inhibited， and it was clear that VacJ promoted the formation of App BF[11].
　　LuxS gene regulation
　　LuxS is a kind of active enzyme. When joining in activating methyl cycle， its byproduct autoinducer 2（AI2） could be taken as densitysensing signals of some bacteria. In porcine respiratory pathogen App， induction effect of LuxS on AI2 has been proved. In App strain of LuxS deletion， the formation and pathogenicity of BF obviously weaken. To fully understand the function of LuxS gene， comparative study on gene expression profiles of App LuxS deletion strain and its parent strain in four growth stages was conducted by gene core technology. The results showed that differential expression of many genes related to infection was found. In early logarithmic growth stage of LuxS deletion strain， BF formation gene pgaABC was upregulated， while 9 genes related to adhesion in latter logarithmic growth stage were downregulated， and related genes joining in iron ion uptake and metabolism in four growth stages were all upregulated. In phenotypic study， AI2 was used to compensate virulence traits of LuxS deletion mutant. The results displayed that LuxS deletion enhanced the formation of App BF， and reduced adhesion ability of bacteria. But AI2 increasing the formation of App BF may not depend on LuxS. LuxS controlled iron ion uptake via the production of AI2. These results revealed that both LuxS and AI2 may play a role in the virulence formation of App[12-13]. 　　Adh gene regulation
　　Adhesion is the crucial first step in infection process. Trimer autotransporting mucin （TAAS） has been identified as new virulence factor of App， but its effect in pathogenicity is known little. Expression in vitro and adhesion test results showed that YadA sample region （Adh） was the optimal adhesive functional area. Additionally， Adh could protect the attacks from App serotype 1， serotype 3 and 5 A strains in induced part of mice. In vitro， Adh gene deletion obviously declined bacterial accumulation， BF formation and the adhesion to host cell. In vivo， Adh deletion strain （5B┝Adh） could defer clinical symptoms of piglets， decrease the generation of inflammatory cytokines， ease lung injury， and significantly decline pathogenicity of piglets. Via gene chip analysis and quantitative PCR， specific gene expression of 5B┝Adh strain and control strain 5b L20 infecting lung tissue of piglets was verified. The results showed that there was differential expression of 495 genes in 5B┝Adh infecting lung tissue （221 genes upregulated and 274 genes downregulated）， and the expression of processed and presented gene IFI30 by the antigen also increased. Therefore， Adh could enhance pathogenicity of bacteria by inhibiting immune recognition of host[14]. It shows that Adh could regulate bacterial accumulation， and formation， adhesion and pathopoiesis of BF.
　　Conclusions
　　App BF belongs to polymers with spatial structure in vitro， and its formation is affected by gene arc and O antigen besides above genes[15-16]. These data show that BF formation is affected by many factors. Wu C et al.[17]found that zinc ion in certain concentration could inhibit the formation of App BF. Interestingly， acellular extract isolated from 5 type App BF of serology could inhibit the formation of BF in S. aureus， Staphylococcus epidermidis and Actinobacillus actinomycetemcomitans， but did not affect the formation of serum 5 type BF. Physical and chemical analysis showed that antiBF activity in the acellular extract was from polymer polysaccharide， while acellular extract isolated from serum 5 type capsular polysaccharide deletion mutant did not show the resistance to BF. The plasmid carrying capsular polysaccharide gene was transformed in above mutants， and the obtained acellular extract could recover the resistance to BF. The purified serum 5 type capsular polysaccharide had the resistance to BF of S. aureus， and the extract of App wild type could not inhibit the growth of S. aureus， but could inhibit the adhesion among S. aureus and the combination with stainless steel surface. Additionally， the extract of App wild type wrapping on the polystyrene surface could resist the formation of BF in S. aureus， but capsuledeficient strain could not resist the formation of BF in S. aureus. Research showed that App serum 5 type capsular polysaccharide could inhibit the interaction among cells and the interaction between cell and other surfaces[18]. App serum 5 type capsular polysaccharide has broadspectrum antiBF activity of nonkilling bacteria. In the future， these antiBF polysaccharides may reveal some new functions of bacterial polysaccharides， which could provide new thinking for developing antiBF preparation in clinic. 　　References
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