Comamonas kerstersii細菌致病性的基因組分析(英文)

Genomic Insights of Pathogenicity of Comamonas kerstersii

WANG Hui¹，MING Desong²，WANG Mingxi¹

（ 1. School of Medicine，Huaqiao University，Xiamen 361021，China；2. Quanzhou First Hospital Affiliated to Fujian Medical University，Quanzhou 362000，China ）

Abstract：To achieve better management of Comamonas kerstersii（C. kerstersii） infections，the aim was to understand its virulence and pathogenicity by analyzing the virulence factor genes （VFGs） in the genome of C. kerstersii 121606. The genome was sequenced previously， and the VFGs were predicted by BLAST searching through the VFDB database. The results showed that C. kerstersii contained a large number of VFGs，some of which were exemplified to correlate to the survival and growth in humans，and the pathogenicity of C. kerstersii in the gastrointestinal tract and lungs. Genomic analysis revealed that C. kerstersii might be an intracellular pathogen. The harbored VFGs were conducive to explaining its virulence and pathogenicity in gastrointestinal tract and lung infections reported in the literature or encountered in clinic. The data also provided ideal targets for novel antibiotic and vaccine development for the therapy of C. kerstersii infections.

Keywords：Comamonas kerstersii；virulence factor gene；pathogenicity；intracellular pathogen；gastrointestinal tract infection；lung infection

CLC Number：中圖分類號：Q 939.48；R 378文獻標志碼：Document Code：A Article Number：文章編號：1000-5013（2024）03-0394-23

摘要：為了更好地管理Comamonas kerstersii （C. kerstersii）感染，通過分析C. kerstersii菌株121606基因組中的毒力因子基因（VFGs）來了解其毒力和致病性。 對已經(jīng)完成測序的C. kerstersii 121606基因組，通過BLAST搜索VFDB數(shù)據(jù)庫來預(yù)測其VFGs。結(jié)果表明：C. kerstersii含有大量VFGs，其中一些與其在人體內(nèi)的生存和生長，以及在胃腸道和肺部的致病性有關(guān)?；蚪M分析表明：C. kerstersii可能是一種細胞內(nèi)病原體。C. kerstersii所攜帶的VFGs有助于解釋其在文獻中所報道的胃腸道感染和臨床工作中所遇到的肺部感染中的毒力和致病性，這些數(shù)據(jù)也為新型抗生素和疫苗開發(fā)提供了理想的靶點，以用于治療C. kerstersii感染。

關(guān)鍵詞：Comamonas kerstersii；毒力因子基因；致病性；細胞內(nèi)病原體；胃腸道感染；肺部感染中圖分類號：

Comamonas kerstersii （C. kerstersii） is a Gram-negative，aerobic，motile rod bacterium^[1]，which is found ubiquitously in water，soil，and plants^[1]. As summarized in our previously submitted work，we knew that，it primarily causes intra-abdominal infections，such as peritonitis and appendicitis，as well as extra-abdominal infections，including lung infection encountered in our clinical settings. Due to difficulties in identification using conventional phenotypic methods or even 16S rRNA gene sequencing，the actual prevalence of its infections are underestimated. While it is rarely resistant to antibiotics and is closely associated with a favorable clinical prognosis，we have also isolated a pan-drug resistant C. kerstersii strain 121606 from an 82-year-old male patient. Given the potential health threat posed by pan-drug resistance，we have conducted genomic studies to elucidate the mechanisms of pan-drug resistance at the genomic level （submitted）. In this article，we aim to further understand the survival ability，virulence，and pathogenicity of C. kerstersii，particularly in the gastrointestinal tract and lungs，by analyzing the virulence factor genes （VFGs） in the genome of the exemplified C. kerstersii 121606 strain. This strain shares nearly identical VFGs with our two other C. kerstersii strains： one （strain 12322-1） isolated from the sputum of a patient with a fatal lung infection，to whom it promoted the patient′s death，and another （strain 202149） isolated from peritoneal pus of a patient with acute perforated appendicitis.

1 Materials and Methods

1.1 Genome Sequencing，Assembly，Annotation for C. kerstersii 121606

These works had been completed as described in previously submitted article. The 16S rRNA gene sequence （1 460 bp，GenBank No. KY014106） and the genome sequences of C. kerstersii strain 121606 （GenBank assembly accession No. GCA_002002445.1，ASM200244v1） had been deposited into GenBank.

1.2 Prediction of VFGs in C. kerstersii 121606 Genome

The VFGs in C. kerstersii 121606 were predicted by conducting a BLAST search against the VFDB protein Set B database^[2] in collaboration with Beijing Novogene Bioinformatics Technology Co.，Ltd.. The identity parameter was set at gt;40%. No stringent cut-off parameters were applied to further filter the initially predicted VFGs because doing so would exclude a large number of potentially unknown VFGs and result in an inability to correlate the remaining VFGs to the infectivity of C. kerstersii in patients.

2 Experimental Results and Analysis

The VFGs inC. kerstersii 121606 were listed in table 1 （discussed） and table 2 （not discussed），such as the genes involved in formation/biosynthesis/regulation of capsular polysaccharide （rmlA，rmlB，rmlC，wbfV/wcvB，wbjD/wecB，wecC，gtaB），LOS （galE，kdsB，kdtA/waaA，lpxA，lpxB，lpxC，orfM），lipopolysaccharide （acpXL，fabZ，gtrB，hisF，kdsA，lpxD，kdtB），motility/attachment apparatuses，fimbriae （cupA1，cupA3，cupD2，cupD4，cupD5），type IV pili （pilA，pilB，pilC，pilD，pilE，pile，pilG，pilH，pilJ，pilM，pilN，pilQ，pilR，pilT，pilT2，pilU，pilZ，rpoN），flagella （motility： flaA/flmA，flmH，fleN，flgB，flgC，flgD，flgE，flgF，flgG，flgH，flgI，flgJ，flhA，flhB，flhC，flhD，fliA，fliC，fliE，fliF，fliG，fliI，fliM，fliN，fliQ，flip，fliR，fliS，motA，motB；chemotaxis： cheA，cheB，cheD，cheR，cheW，cheY，cheY1，tsr，tar/cheM），yersiniabactin siderophore （fyuA，ybtQ，ybtP，irp2，ECVR50_2112），Haemophilus iron transport system HitABC （hitA，hitC），molecular chaperones （groEL，dnaK），hemolysin （hlyⅢ，hlyA），heme uptake system （hutC，hutD，bhuT，hmuV，phuS），HasA-type hemophore-mediated heme uptake system （hasD，hasE），heme biosynthesis pathway （hemB，hemC，hemE，hemH，hemL，hemN），heme utilization （AB57_0992），the ATP-binding cassette （ABC） transporter-FbpABC （fbpC） and other iorn uptake systems （fatD，vctC，irgA，fur）.

Several conclusions can be drawn from table 1：

1） C. kerstersii possesses multiple iron acquisition systems vital for the bacterial survival and virulence.

（a） The yersiniabactin siderophore system sequesters irons from transferrin and lactoferrin and delivers them to the cytosol and is an essential virulence factor；the yersiniabactin siderophore system also acts as chelator of Cu²⁺，Zn²⁺，Ni²⁺ and plays the corresponding important role in bacterial survival in enviroment and virulence in human；

（b） Haemophilus Iron Transport system functions in assimilation of iron from human transferrin；

（c） FbpABCtransporter functions in the periplasm-to-cytosol transport of iron；

（d） Molecular chaperones are involved in iron uptake；

（e） Hemolysins lyse erythrocyte cells and release heme into blood and act as vital virulence factors；

（f） Heme uptake pathways；

（g） Heme biosynthesis pathway.

2） C. kerstersii possesses many virulence factors crucial for their growth，survival and pathogenicity in gastrointestinal tract.

（a） Type IV pili are essential for bacterial colonization and pathogenicity in gastrointestinal tract；

（b） Flagella are vital for bacterial pathogenicity to gastrointestinal tract；flagella are involved in the motility and chemotaxis functions in intestinal colonization and pathogenicity in bacterial gastroenteritis；flagellar structure，assembly，biosynthesis regulation，gene clustering；constituents and the signal pathway of flagellar chemotaxis；

（c） The virulence factors urease，SodB，DnaK，Elongation factor Tu vital for bacterial growth，survival and pathogenicity in the gastrointestinal tract.

3） The virulence factors Mip，ClpP，ClpC，DnaK，GroEL，MgtC and Icl are vital for intracellular survival and infectivity of C. kerstersii to lung.

3 Discussion

As described above，C. kerstersii mainly causes intra-abdominal infections，and the gastrointestinal tract is its natural habitat for survival and virulence. Since we also isolated a C. kerstersii strain （12322-1） from the sputum of our patient，it is possible that the respiratory tract is also a habitat for its survival and virulence. The correlation of VFGs with bacterial survival，growth in humans，and infectivity of C. kerstersii in the gastrointestinal tract and lungs was exemplified by analyzing the following VFGs.

3.1 C. kerstersii Possesses Multiple Iron Acquisition Systems Vital for Bacterial Survival and Virulence

3.1.1 Iron Sources in Host are Vital for Bacterial Survival

Iron is essential for almost all organisms^{[3- 4]}. In the human body，iron exists as ferric （Fe³⁺） and ferrous （Fe²⁺） ions，as well as iron-heme coordinated with protoporphyrin IX^[5]. The concentrations of free iron （mostly Fe²⁺） and insoluble iron （Fe³⁺，with extremely low solubility of 10^-18 mol·L^-1） are negligible^[5-6]. Heme-iron （Fe²⁺） incorporated into hemoglobin （Hb） in red blood cells is the main form of circulating iron，accounting for approximately 80% of all human iron^[5]. The main circulating non-heme iron in the human bloodstream includes transferrin （Fe³⁺） and lactoferrin （Fe³⁺）^[5]. C. kerstersii has several iron acquisition mechanisms，which may confer higher survival ability and pathogenicity^[7].

3.1.2 Yersiniabactin Siderophore System Sequesters Irons From Transferrin and Lactoferrin and Delivers Them to Cytosol and is an Essential Virulence Factor

The Yersiniabactin siderophore is made up of one salicylate，one thiazoline，and two thiazolidine rings^[4]. It has an extremely high affinity for ferric （Fe³⁺） ions （K_a=4×10^-36" L·mol^-1）^[8]. Yersiniabactin efficiently removes Fe³⁺ ions from transferrin and lactoferrin^[4]，then binds to the TonB-dependent outer membrane receptor-Psn in Yersinia pestis^[4] and FyuA in Y. enterocolitica^[9] to pass through the outer membrane into the periplasm^[4]. It subsequently delivers Fe³⁺ ions to the cytosol via the inner membrane ABC transporter，which is composed of two inner membrane fused-function permease/ATP-binding proteins，YbtP and YbtQ^[4]，followed by the reduction of Fe³⁺ to Fe²⁺.

The yersiniabactin siderophore promotes the growth of Y. enfevocolitica and Escherichia coli under iron-restricted conditions^[9]. It is also a virulence factor for other highly pathogenic bacteria，such as Y. pseudotuberculosis，Y. enterocolitica biotype IB，and extraintestinal pathogenic E. coli （ExPEC）^[10]. Additionally，the yersiniabactin siderophore-based iron transport system is essential for the pathogenesis of bubonic and pneumonic plague^[4，11]，which is a flea-borne zoonosis caused by Y. pestis^[12].

Yersiniabactin is synthesized througha nonribosomal peptide/polyketide mechanism involving several proteins，including high-molecular-weight protein 1 （HMWP1），HMWP2，YbtD，YbtT，YbtE，YbtS，and YbtU. Its secretion requires an inner membrane exporter encoded by a four-gene operon （ybtPQXS）^[4]. Most of the genes required for yersiniabactin biosynthesis （ybtT，ybtE，ybtS），transport （fyuA，ybtP，ybtQ） and the regulation （ybtA） are located within a Yersinia high-pathogenicity island （HPI）^[4，13]，This island is also crucial for the virulence of uropathogenic E. coli （UPEC），the main causative agents of non-nosocomial urinary tract infections，particularly in the highly inflammatory infection environment of high-titer mouse cystitis^[14].

C. kerstersii possesses the yersiniabactin siderophore system，as it ontains genes encoding for the yersiniabactin siderophore system： （a） fyuA；（b） ybtQ/ybtP，which are overlapping genes，with each encoding half of an inner membrane ABC transporter containing an amino-terminal membrane-spanning permease domain and a carboxy-terminal ATPases domain^[15]；and （c） irp2，which encodes the putative peptide synthetase-HMWP2 for the siderophore yersiniabactin^[16]. Laboratory verification of the presence of these yersiniabactin biosynthesis genes contained in HPI in C. kerstersii genome would help clarify its pathogenicity to patients.

3.1.3 Haemophilus Iron Transport System Functions in Assimilation of Iron From Human Transferrin

C. kerstersii may be able to utilize another classic high-affinity，TonB-independent iron acquisition system-called Hit （Haemophilus Iron Transport） ABC. This system is composed of a periplasmic iron-binding protein （HitA），a cytoplasmic permease （HitB），and a nucleotide-binding protein （HitC） found in Haemophilus influenza^[17]. These components could potentially allow C. kerstersii to assimilate iron from human transferrin，as hitA and hitC were predicted in the C. kerstersii 121606 genome.

3.1.4 FbpABC Transporter Functions in Periplasm-to-Cytosol Transport of Iron

Together with hitABC，the prediction of fbpC in the C. kerstersii 121606 genome suggests that it has an additional periplasmic protein-dependent iron ABC transporter，F(xiàn)bpABC. This transporter consists of the ferric binding protein （FbpA），a homologue of HitA，F(xiàn)bpB，a cytoplasmic permease，and FbpC protein，a nucleotide binding protein^[18]. It is required for iron uptake from the host ferric binding proteins^[19].

Additionally，C. kerstersii 121606 contains： （a） iron compound ABC transporter permease protein gene，fatD；（b） Fe³⁺ ABC transporter ATP-binding protein gene，vctC；（c） iron-negatively regulated virulence gene，irgA，and d） its negative regulator gene，fur^[20]. Fur was reported to sense intracellular iron amount and maintain iron homeostasis in Bacillus subtilis^[21]. Fur might also be regulator of hitA as it was a regulator of afuA，which encodes a homologue of HitA^[22].

3.1.5 Molecular Chaperones are Involved in Iron Uptake

Helicobacterpylori is the major pathogen of gastritis^[23] and requires iron for growth^[24]. Bacterial GroEL，a homologue of human heat shock protein 60 （Hsp60），is a well-characterized molecular chaperone that prevents aggregation and facilitates proper protein folding in bacteria^[25]. It，along with urease （discussed below），are the major surface-exposed proteins of H. pylori^[26]. GroEL can assist in iron acquisition for H. pylori through specific binding with iron extracellularly and scavenging iron from heme^[27]，as well as in the maturation of metalloenzymes by supporting the insertion of a metal cluster into an apoprotein，such as Molybdenum-iron protein^[28]. GroEL and another bacterial molecular chaperone，DnaK，the main homologue of human Hsp70 in bacteria^[29]，were also shown to bind to lactoferrin^[30]. The prediction of groEL and dnaK in C. kerstersii 121606 genome indicated that this bacterium had these iron acquisition mechanisms.

3.1.6 Hemolysins Lyse Erythrocyte Cells and Release Heme Into Blood and Act as Vital Virulence Factors

C. kerstersii 121606 possesses the genes （hlyⅢ，hlyA） that encode hemolysins capable of lysing erythrocyte cells，releasing Hb into the blood serum，and converting it into oxidized Hb （metHb），in which ferric heme is loosely bound and can be utilized by bacterial heme uptake systems^[5]. It has been reported that haemolysin Ⅲ （encoded by hlyⅢ） from B. cereus can form 3–3.5 nm transmembrane pores in diameter on the erythrocyte membrane and lyse them^[31]. HlyA （110 ku，listeriolysin O）^[32] can also lyse erythrocytes^[33] and a wide spectrum of cells，such as granulocytes，monocytes，and endothelial cells^[33]. When produced by the intramacrophage Listeria monocytogenes，a food-borne pathogen causing listeriosis with a mortality rate of approximately 20%^[32]，HlyA can break the endosome membrane and help this bacterium escape the hostile endosome environment^[34]. HlyA is verified as a unique and essential virulence factor for virulent L. monocytogenes^[32] and plays a vital role in E. coli-caused extraintestinal diseases^[35].

3.1.7 Heme Uptake Pathways

These findings of the genes hutC，hutD，bhuT，hmuV in C. kerstersii 121606 indicated that C. kerstersii had multiple heme uptake systems.

Gram-negative bacteria are found to have two heme uptake mechanisms^[5] to transport of host heme，heme proteins （Hb，hemopexin） into cytosol，one is through the heme scavenging proteins-hemophores，another is by binding outer membrane receptors，then heme is transported across the bacterial outer membrane via a TonB-dependent outer membrane receptor into the periplasm，then transferred to the periplasmic heme binding protein （PBP），which transmits heme to the inner membrane ABC transporter complex，finaly translocated into the cytoplasm^[5，36-37].

The prediction of the genes，hutC，encoding a putative inner membrane permease，and hutD，encoding a putative ABC-transporter ATPase，which form the heme/Hb ABC transporter-hutCD^[36-38] in C. kerstersii 121606，and bhuT，encoding the periplasmic heme-binding protein which functions as HutB^[37]，indicated that they might own the TonB dependent heme uptake mechanism^[36]，but whether another component-the outer membrane heme receptor encoding gene hutA is in C. kerstersii strains requires experimental evidence.

The presence of hmuV in C. kerstersii 121606，which encode a ATPase，suggested the possible presence of another heme ABC transport system-HmuTUV，if the genes encoding other two components-the periplasmic binding protein （hmuT），and the permease （hmuU） are verified to be present in C. kerstersii by experiment^[39].

C. kerstersii may also possess HasA-type hemophore-mediated heme uptake system，a two component of a ABC exporter-HasDEF，which exports a heme acquisition protein，HasA^[40]，as C. kerstersii 121606 contained the genes hasD，encoding ABC transporter ATP-binding protein，and hasE，encoding ABC transporter periplasmic substrate-binding protein^[40].

3.1.8 Heme Biosynthesis Pathway

In addition to heme uptake，C. kerstersii may also have the ability to biosynthesize heme. The heme biosynthetic pathway starts from GltX-catalyzed conversion of L-glutamate to charged glutamyl-tRNAGlu，which acts as substrate of glutamyl-tRNA reductase （HemA），the latter works sequentially with glutamate-1-semialdehyde aminotransferase （HemL） to synthesize 5-aminolevulinic acid （ALA）. Subsequently，ALA is used to synthesize uroporphyrinogen Ⅲ by porphobilinogen synthase （HemB），then heme by uroporphyrinogen decarboxylase （HemE） and protoporphyrin ferrochelatase （HemH）^[41]. Prediction of the genes endcoding HemB，HemC （porphobilinogen deaminase），HemE，HemH （ferrochelatase），HemL and HemN （coproporphyrinogen Ⅲ oxidase） in C. kerstersii 121606 suggests that it possesses this heme biosynthesis pathway^[42].

3.1.9 Iron Acquisition Proteins are Potential Drug and Vaccine Targets

The iron acquisition proteins play an essential role in the pathogenesis of causative bacteria. They have been identified as potentially novel antibiotic and vaccine targets to combat infections. For example，F(xiàn)yuA^[43]，HitA^[44]，GroEL and DnaK^[45] are the potential vaccine candidates against bacteria.

3.2 Yersiniabactin Siderophore System Also Acts as Chelator of Cu²⁺，Zn²⁺，Ni²⁺ and Plays Corresponding Important Role in Bacterial Survival in Enviroment and Virulence in Human

Yersiniabactin is also a chelator of Cu^{2+ [46]}，Zn^2+[16]. Copper serves as both a nutrient and a toxin during bacterial infections. Yersiniabactin efficiently scavenges extracellular Cu²⁺ in the form of a Cu²⁺-Yersiniabactin complex. It then uses the same Fe³⁺-Yersiniabactin transport proteins to facilitate copper import and release in UPEC. This supports copper-dependent enzyme activity in low copper conditions，such as cuproenzymes^[47]，and helps UPEC resist copper toxicity in higher copper conditions^[47] by preventing Cu²⁺ reduction to the more bactericidal Cu^+[46]. Additionally，it protects UPEC against phagocytic killing by catalyzing superoxide dismutation intraphagocytically with the superoxide dismutase （SOD）-like activity of the Cu²⁺-Yersiniabactin complex^[46].

Zn²⁺ is a necessary micronutrient during bacterial infections. The yersiniabactin siderophore also plays a crucial role in acquiring Zn²⁺ and causing lethal pathogenesis in mouse pneumonic plague^[16]. In addition to Fe³⁺，Cu²⁺，and Zn²⁺，yersiniabactin also has a high affinity forchelating Ni²⁺，Cr³⁺，Co³⁺，and Ga^{3+ [14]}，as well as Co²⁺ and Pd^2+[48]. This promotes the survival of bacteria in heavy metal-polluted environments.

3.3 C. kerstersii Possesses Many Virulence Factors Crucial for Their Growth，Survival and Pathogenicity in Gastrointestinal Tract

3.3.1 Type IV Pili are Essential for Bacterial Colonization and Pathogenicity in Gastrointestinal Tract

As proteinaceous hair-like appendages，pili are essential for bacterial adherence to human gut cells^[49]. Type IV pili （T4P），which are approximately 5-8 nm in diameter and several micrometers in length，have three subtypes： Type IVa pili （T4aP），Type IVb pili （T4bP），and Type IVc pili （T4cP）^[50]. They are predicted to be produced by about 30% of the known gut microbiome^[49]. T4P promotes the intestinal colonization of enterohemorrhagic E. coli （EHEC） O157：H7，a food-borne pathogen of hemorrhagic colitis^[51]，enterotoxigenic E. coli （ETEC），the most common bacterial pathogen of diarrhea in developing countries and traveler’s diarrhea^[52]，and enteropathogenic E. coli （EPEC）^[53]. It also promotes the survival，virulence，and transmission of EHEC O157：H7^[51]，invasion of epithelial cells，hemagglutination of erythrocytes，biofilm formation，and twitching motility of EHEC O157：H7^[51].

The biogenesis process of Type IV pili in Gram-negative bacteria begins with the attachment of an ATPase-PilB to the cytoplasmic ring formed by PilM and the platform protein PilC^[49]. PilM，along with the subunits PilN，PilO，and PilP，forms an inner membrane alignment complex called PilMNOP. This complex has a cage-like ring in the inner membrane and periplasm，which is formed by conformational changes in PilN and PilO resulting from the attachment of PilB to PilM. Then，the platform protein PilC incorporates the major pilin subunit PilA after the removal of the leader peptide by the prepilin peptidase PilD. PilD also methylates PilA or minor pilins，such as pilE，into the growing pilus based on the PilMNOP alignment complex. Afterward，PilA is translocated through the outer membrane pore formed by PilQ to elongate the pilus^[49]. The cytoplasmic ATPase PilB drives pilin assembly，while the PilT ATPase drives disassembly or retraction. Both of them can reversibly associate with the PilC protein^[49]. In the final step，the minor pilins are synthesized in a similar way to PilA and create a priming complex for pilin formation at the pilus tip and along the pilus^[49].

The presence of the Type IV pili biogenesis genes encoding PilA，PilB^[54]，PilC，PilD，PilE，PilJ {a minor pilin and a methyl-accepting chemotaxis protein （Mcp）-like chemosensor，which directly interacts with the major pilin subunit PilA and regulates surface-induced gene expression and pathogenicity^[54]}，PilG {a polytopic membrane protein dedicated to assist transformation of bacteria by binding DNA and interacting with the N-terminal region of PilQ，which can also bind DNA^[55] and essential for twitching motility）^[56]}，PilH {an ABC transporter homologue required for the biogenesis of any bacterial pilus type^[57]}，PilM，PilN，PilQ {a secretin working in concert with PilG to bind DNA during T4P-mediated transformation^[55]}，PilR {the response regulator of a two （PilS and PilR）-component transcriptional regulatory system dedicated to activate transcription of pilA^[58]}，PilT，PilT2，PilU {also a retraction ATPase motor^[54]}，PilZ {which can stimulate T4P formation or regulate T4P-dependent motility）^[59]} in C. kerstersii 121606 indicated that C. kerstersii possessed the virulence factor T4aP possibly invovled in the pathogenicity of C. kerstersii in intesintal diseases^[49]，such as peritonitis，appendicitis，as mentioned above.

3.3.2 Flagella are Vital for Bacterial Pathogenicity to Gastrointestinal Tract

3.3.2.1 Flagella are Ivolved in Motility and Chemotaxis Functions in Intestinal Colonization and Pathogenicity in Bacterial Gastroenteritis

Flagella are approximately 20 nm in diameter^[60]. The possession of rapid，darting motility in viscous milieus conferred by the polar flagella is an exclusive characteristic of virulent Campylobacter jejuni and E. coli^[61]，the major pathogen of acute human bacterial gastroenteritis worldwide^{[62- 63]}，and are required for intestinal mucus colonization^[61]，a key pathogenicity determinant in intestinal infection by C. jejuni^[64-65] and H. pylori^[66]. Additionally，they function in secreting virulence proteins，microcolony formation，biofilm formation and escape of the innate immune response^[61]. Chemotaxis conferred by polar flagella is also critical for colonization and pathogenicity in the establishment of gastrointestinal infections by C. jejuni^{[61- 62]} and H. pylori^[66].

3.3.2.2 Flagellar Structure，Assembly，Biosynthesis Regulation，Gene clustering

Flagella are composed of approximately 20 000-30 000 protein subunits，consisting of over 20 different types of proteins^[67]. They comprise： （a） the base located in the cytoplasm and inner membrane，which is composed of the flagellar type Ⅲ secretion system （T3SS），a membrane-embedded export gate composed of six transmembrane proteins-FlhA，F(xiàn)lhB，F(xiàn)liO，F(xiàn)liP，F(xiàn)liQ，and FliR within the central membrane patch of the ring，the water-soluble ATPase ring complex composed of three cytoplasmic proteins，F(xiàn)liH，F(xiàn)liI，and FliJ^[68-71]，the inner membrane MS ring （a homomultimer of flagellar motor switch protein FliF）^[68，72]，the cytoplasmic C ring or switch complex {which is formed by two motor/switch proteins FliM and FliN^[68，73] and functions as the flagellar switch and also aids in secretion^[66]}，and the motor {formed by multimers of the motor/switch protein FliG^[68，72]} attached to MS ring^[68]；（b） the coaxial periplasmic rod {a helical cylinder cotaining four flagellin proteins-FliE，F(xiàn)lgB，F(xiàn)lgC，and FlgF in the proximal part and FlgG in the distal part^[66-67]} and the associated ring structures；（c） the surface-localized hook structure （a helical assembly of about 120 FlgE subunits） spanning the periplasm and outer membrane；and （d） the extracellular filament {composed primarily by the major flagellin，F(xiàn)laA，with the minor flagellin，F(xiàn)laB^{[61，66]} or FliC^[67]}. The rod is surrounded by： （a） three disk structures，consisting of the basal （composed of FlgP），the medial paralyzed flagellum protein A （PflA），and the proximal disk （composed of PflB）；（b） the motor components of the base including MotA and MotB （encoded by motA，motB），which forms a proton channel^[74]；（c） the stators of flagellum in periplasm that interact with FliG and function to link proton flow and generation of flagellar rotation torque^{[66，68，72]}；（d） the periplasmic P ring （FlgI） in peptidoglycan；and （e） the L ring （FlgH） in outer membrane^[66，68].

Flagellar assembly begins at the base，then progresses to the hook，and finally to the filament^[70，75]. The peptidoglycan-hydrolyzing activity of the FlgJ protein （peptidoglycan hydrolase） is necessary for flagellar rod formation^[76]. To assemble the hook and filament from the cell surface，their component proteins are transported from the cytoplasm to the distal end of the growing structure by T3SS^[66]. Chaperones FlgN，F(xiàn)liS，and FliT assist in the transfer of flagellin subunits to T3SS from the cytoplasm^[69]. The initial entry of the flagellin subunits into the narrow pore of the export gate is facilitated by the ATPase ring complex and powered by proton motive force，which is controlled via the specific interaction of FlhA with FliJ^[69]. The scaffolding protein FlgD is also essential for flagellar hook assembly in Salmonella typhimurium^[77]. Its export requires FliE，a flagellar basal body protein，and an adaptor protein located between the MS ring and rod substructures^{[68，78]}. The flagellar number is controlled by the antiactivator FleN through binding to the enhancer of the flagellar gene，fleQ，a multidomain sigma-54 （σ⁵⁴） factor^[79].

Flagellar biogenesis is tightly controlled by the activities of σ⁵⁴ and σ^28[66]. For instance，the two-component signal transduction system，F(xiàn)lgSR，is composed of the FlgS sensor kinase and the FlgR response regulator. This system recognizes a regulatory checkpoint in the MS ring and rotor to activate the σ⁵⁴-dependent expression of flagellar rod and hook genes，and ultimately the σ²⁸-dependent flagellins and fed gene expression^[72].

In the genome，a number of flagellar genes are structurally organized as operons，with a master and several secondary levels of gene expression regulation^[80]. Examples include the flgGHIJKL operon^[81]，the flgBCDEF operon，whose genes are transcribed as a single mRNA in a σ⁵⁴-dependent way in Rhodobacter sphaeroides^[82]，the flhBAE operon in Y. enterocolitica^[83]，the flmA（flaA）B operon，and the flmGH operon in Caulobacter crescentus^[84]. Another example of a master regulator in flagellum biogenesis is the flhD operon，which encodes two genes，flhD and flhC in E. coli. This operon produces the FlhD/FlhC complex，which is a transcriptional activator required for the transcription of the three class II operons-fliA，flhB，and fliL^[85]. Additionally，as a subunit of RNA polymerase and a homologue of σ²⁸，the FliA protein activates flagellin synthesis by transcribing the fliC gene^[86].

The prediction of the flagellar components and biogenesis genes in C. kerstersii 121606 indicates that C. kerstersii possesses flagella with intact motility function.

3.3.2.3 Constituents and Signal Pathway of Flagellar Chemotaxis

The signal transduction proteins of chemotaxis include the core components. These consist of chemoreceptors，which sense environmental stimuli and transduce this signal to mediate rotation of flagella. The two-component system is composed of the histidine kinase CheA and its cognate response regulator CheY^[87]. The phosphorylated form of CheY，regulated by ligand binding to the chemoreceptors，interacts with the FliM component of the flagellar motor switch to cause clockwise motor rotation^[88]. The CheW coupling protein physically conjugates chemoreceptors to CheA^[66]. Other components include the phosphatases CheZ and FliY，which promote CheY dephosphorylation，the chemoreceptor modification enzymes CheR （a methyltransferase that transfers methyl groups from S-adenosylmethionine to glutamate residues on the cytoplasmic domains of the chemoreceptors Mcps），and CheB （a methylesterase），that governs adaptation responses to sustained ligand levels by methylating or demethylating glutamyl residues of chemoreceptor respecitively^[66]. The chemoreceptor glutamine deamidase CheD deamidates the Mcps essential for chemoreceptors to effectively transduce signals to the CheA kinase in B. subtilis^[87] and increases the receptor kinase activity or enhances CheC phosphatase activity to regulate the CheY levels in B. subtilis^[89]. Some bacteria have multiple homologues of bacterial chemotaxis and chemosensing genes，such as tsr，tar/cheM，and have a great ability to respond to a wide variety of compounds，like Chromobacterium violaceum^[74]，an extremely virulent opportunistic pathogen ubiquitously distributed in water and soil^[90].

The prediction of cheA，cheB，cheD，cheR，cheW，cheY，cheY1，tsr and tar/cheM in our C. kerstersii 121606 suggested that their flagella have full chemotaxis function.

As a whole，the prediction of almost all of the flagellar components，biogenesis，motility，and chemotaxis genes in C. kerstersi 121606 indicates that they have fully functional flagella，which contributes to their high pathogenicity in the gastrointestinal tract.

3.3.3 Virulence Factors Urease，SodB，DnaK，Elongation Factor Tu Vital for Bacterial Growth，Survival and Pathogenicity in Gastrointestinal Tract

Survival，colonization and pathogenicity of H. pylori in gastric acidic environment depends on the abundantly produced nickel-dependent urease，which hydrolyses urea into ammonia to create a pH neutral microenvironment^[26] and block the phagosome-lysosome fusion to resist against the host immune system^[91]. Most bacterial ureases are made of a （αβγ）₃ （encoded by ureA，ureB，ureC） trimer^[92]. Urease also requires different sets of accessory proteins，such as UreE，UreF，UreG，UreH and UreI^[93] or UreD，UreE，UreF and UreG^[94-95]，to coordinate nickel ions into the UreABC subunits. Considering that the urease encoding genes predicted through VFDB database might be not complete，we manually searched the annotated genome of C. kerstersii 121606，and found that it contained the genes encoding the subunit α，β，γ，and the accessory proteins UreD，UreE，UreF，UreG，meaning that C. kerstersii could express functional urease.

The survival，colonization，and pathogenicity of H. pylori in the gastric acidic environment depend on the abundantly produced nickel-dependent urease. This enzyme hydrolyzes urea into ammonia to create a pH-neutral microenvironment^[26] and blocks the phagosome-lysosome fusion to resist the host immune system^[91]. Most bacterial ureases are composed of a （αβγ）₃ trimer （encoded by ureA，ureB，ureC）^[92]. Urease also requires different sets of accessory proteins，such as UreE，UreF，UreG，UreH，and UreI^[93]，or UreD，UreE，UreF，and UreG^[94-95]，to coordinate nickel ions into the UreABC subunits. Considering that the urease-encoding genes predicted through the VFDB database might not be complete，we manually searched the annotated genome of C. kerstersii 121606 and found that it contained the genes encoding the subunits α，β，γ，and the accessory proteins UreD，UreE，UreF，UreG，indicating that C. kerstersii could express functional urease.

SodB，an iron-cofactored superoxide dismutase，is necessary for H. pylori to combat the oxidative stress produced by neutrophils and macrophages^[96] while colonizing the host gastric mucosa^[97].

DnaK is found on the cell surface ofseveral pathogens，such as human probiotic intestinal microbiota Lactobacillus salivarius，and Bifidobacterium animalis subsp. lactis^[98]. It can be upregulated by bile salts in the gastrointestinal tract of mammals to enhance the adaptation and colonization of B. animalis subsp. lactis in the gut bile environment by acting as a high affinity receptor of host plasminogen^[98].

The elongation factor thermal unstable Tu （EF-Tu，encoded by tuf） is a translational GTPase （G protein），that delivers aminoacyl-tRNAs （aa-tRNAs） to the A-site of the ribosome^[99] and has additional adhesive moonlighting functions^[100]. EF-Tu on bacterial cell surfaces can bind to membrane receptors and fibronectin，a key component of the extracellular matrix （ECM） proteins on the surface of host cells^[100]. It can also bind to human intestinal cells and mucins，indicating the important role of EF-Tu in gut colonization for the probiotic bacterium，L. johnsonii ^[101]. The secreted EF-Tu from H. pylori also promotes adherence and invasion to host cells during pathogenesis^[102].

The above analyses collectively indicate the potential of survival and pathogenicity of C. kerstersi in the gastrointestinal tract. These data help to explain the fact that nearly all reported C. kerstersi infections are gut diseases.

3.4 Virulence Factors Mip，ClpP，ClpC，DnaK，GroEL，MgtC and Icl are Vital for Intracellular Survival and Infectivity of C. kerstersii to Lung

Legionella pneumophilais a Gram-negative，facultative intracellular bacterium^[103] and the causative agent of Legionnaires’ disease，which presents as pneumonia^[104]. It is able to survive and multiply in lung macrophages^[103] and Type I and II pneumocytes^[105]. The Legionella Mip （macrophage infectivity potentiator） protein，a homodimeric lipoprotein of 24 ku located on the bacterial surface and on host membranes^[106-107]，is necessary for the early intracellular infection step of L. pneumophila to alveolar macrophages^[108] and the human phagocytic cell line U937^[109]，intracellular multiplication^[110]，and the full virulence of L. pneumophila in the lungs of guinea pigs and the spread to the spleen after intratracheal inoculation^[108]. It also facilitates L. pneumophila to pass through the collagen IV-containing ECMs or lung epithelial cell lines，and disseminate within the lung tissue and spread to the spleen^[111].

ClpP is a 21.6 ku serine-type protease in E. coli that is highly conserved in prokaryotes and eukaryotes^[112]. It plays a similar role in bacterial pathogenicity in the lungs. ClpP is necessary for the colonization of Streptococcus pneumoniae in the nasopharynx，survival in the lungs of mice after intranasal challenge，and intracellular survival in murine macrophages^[113]. ClpP must bind with the Hsp100/Clp family of molecular chaperones，ClpA，or ClpX，or ClpC，to form the Clp protease ClpAP，ClpXP，or ClpCP complex in order to degrade aggregated protein^[112]. Therefore，it is understandable that ClpC ATPase is also found to promote the intracellular survival of the facultative intracellular pathogen L. monocytogenes in macrophages and survival in host tissues^[114].

Similar to the ClpC ATPase，two other chaperones，DnaK and GroEL，play important roles in the survival and pathogenicity of bacteria. The stress-induced DnaK homologue-Hsp70 is involved in protein folding，proteostasis control^[29]，and removal of aggregated proteins^[115]. It has been verified to be essential for intramacrophage survival of the intracellular pathogen S. enterica serovar Typhimurium，invasion of epithelial cells，leading to systemic infection^[116]，and intramacrophagic replication of Brucella suis^[117].

MgtC is also a vital virulence factor for several intracellular pathogens，such as Burkholderia cenocepacia^[118]，M. abscessus^[119]，both of them being important respiratory tract pathogens in patients with cystic fibrosis，Mycobacterium tuberculosis^[120]，S. enterica serovar Typhimurium^[121]，and Bordetella pertussis，the causative agent of whooping cough^[122]. It also promote bacterial survival of B. cenocepacia^[118]，M. abscessus^[119]，and M. tuberculosis^[120] within macrophages through postponing the phagolysosomal fusion of bacteria^[118]. These functions of mgtC are also observed in P. aeruginosa，a highly lethal extracellular pathogen in cystic fibrosis patients with persistent pulmonary infections^[123]，during the intramacrophage phase or when there is a limitation of Mg²⁺，impairing host phagocytic functions^[124].

Isocitrate lyase （Icl），an enzyme indispensible for the metabolism of fatty acids，has been to promote the persistence of M. tuberculosis in activated macrophages and its virulence in mice^[125]. It is also indispensable for the pathogenesis of Pseudomonas aeruginosa in the rat lung infection model^[126].

These predicted VFGs in C. kerstersii 121606 indicate that C. kerstersii might be an intracellular pathogen，especially forr lung macrophages and Type I and II pneumocytes. This helps to explain the infectivety of C. kerstersii in our patient′s lung.

In summary，these exemplified VFGs helped us understand the ability of intracellular survival，growth in humans，and the infectivity of C. kerstersii in the gastrointestinal tract and lungs of the reported cases and our patients. Some of them might act as novel drug and vaccine targets.

4 Conclusion

C. kerstersii possessed a wide range of VFGs to demonstrate its status as an intracellular pathogen and to elucidate its pathogenicity in the reported gastrointestinal tract infection and a case of lung infection encountered in our clinical work. These findings also offer potential targets for antibiotic innovation and vaccine development to address the rising incidence of C. kerstersii infections.

Acknowledgment：We express gratitude towards the Beijing Novogene Bioinformatics Technology Co.，Ltd.，for providing technical support.

Ethics Statement：This article does not contain any studies with humans or animals performed by any of the authors. This work was approved by the Ethics Committee of Quanzhou First Hospital.

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