LIANG Hai-yun, Li Qi,2,3, HUANG Li-ya, WANG Li-fang, ZHOU Xiang-dong,2,3?

1.Department of Respiratory Medicine, First Affiliated Hospital of Hainan Medical College, Haikou 570102, China

2.Key Laboratory of Tropical Disease Prevention and Control, National Health Commission, Hainan Medical College, Haikou 570102,China

3.Hainan Respiratory Disease Center, Haikou 570100, China

Keywords:

ABSTRACT Burkholderia pseudomallei is the pathogen that causes melioidosis.Melioidosis has a long duration of chronic infection, atypical clinical manifestations at acute onset, and is prone to life-threatening complications and poor prognosis.Understanding the pathogenesis and drug resistance mechanism of Burkholderia pseudomallei will effectively help the diagnosis and treatment of the disease and improve the prognosis.This review focuses on the extracellular movement of Burkholderia pseudomallei in host cells, the way of infecting host cells, virulence factors, and drug resistance mechanisms (efflux pumps, changes in target sites, etc.).This study provides a possible direction for the early diagnosis, treatment and control of melioidosis caused by this bacterium.

Melioidosis is an infectious disease caused by Burkholderia pseudomallei, which is a zoonotic disease, and the incidence area is generally between 20°north and 20°south latitude, mainly distributed in Hainan, Guangxi, Guangdong, Fujian, etc.[1,2].The infection is mainly caused by contact with contaminated soil or water.Skin contact and inhalation are the main routes of infection, and can also be transmitted to the infant through mother’s breast milk[3].This disease is generally sporadic without obvious seasonality, but the incidence is higher in rainy season, flood and typhoon days[4, 5].People are generally susceptible to melioidosis,and the incidence rate is high among farmers who have contact with land, diabetes patients and people with low immunity, among which type 2 diabetes has been confirmed as an independent risk factor for melioidosis[6].

Patients with melioid infection have no specific clinical manifestations, and the infection can involve multiple systems, so it is also called “great mimicker”.85% result in acute infections,11% in chronic infections lasting months to years, and 5% in latent infections.Acute infections such as melioidosis pneumonia and septicemia due to melioidosis are common, often accompanied by systemic multivisceral abscesses (75% in the spleen and 45%in the liver), and are often complicated by sepsis or septic shock in critically ill patients[7].The imaging changes of pulmonary infection are similar to those of cavity tuberculosis, lung abscess,and pneumonia caused by highly virulent Klebsiella pneumoniae[8].The early missed diagnosis and misdiagnosis rate in non-epidemic areas is high.The natural drug resistance mechanism and immune escape of Burkholderia melioides make it resistant to a variety of antibiotics[9], and the disease progresses rapidly.

Cause of the above-mentioned characteristics of melioidosis,it is particularly important to fully understand the pathogenic mechanisms and drug-resistance mechanisms of melioidosis in the future.In this paper, we discuss the pathogenic and drug-resistance mechanisms of Burkholderia pseudomallei, with a view to providing insights into disease diagnosis, treatment, and the development of vaccines.

1.Mechanisms of extracellular movement of Burkholderia pseudomallei

Burkholderia melioides is a soil saprophytic bacterium, which is gram-negative, opportunistic and facultative intracellular parasite.It has flagella, no buds, no capsule, aerobic, and grows well on ordinary medium[10].Thomas et al.conducted a colonization experiment of melioidosis on model mice and found that bacteria in small droplets (1 μm) colonized the epithelium of the lower respiratory tract, while bacteria in large droplets (12 μm) were mainly associated with nasal mucosa and nasal associated lymphoid tissue (NALT)[11].However, regardless of the initial colonization site, Burkholderia melioid infection spreads rapidly throughout the body within 72 h, with colonization visible in distal organs.However, Burkholderia melioides migrates outside host cells and adheres to host cells mainly depending on the following structures:

1.1 Flagellum

The flagellum of bacteria not only allows bacteria to move in the environment, but in many species it is required for invading host cells.The flagellin subunit of B.melioides is encoded by FLIC(bpsl3319).Deletion or inactivation of this gene has resulted in non motile strains in experiments[12-15].Inglis et al found that a mutant strain of B.melioides lacking flagella was unable to attach to the trophozoites of Acanthamoeba[16], considering that flagella allow it to adhere to cells early.And may function as natural antigens.

1.2 Type IV pili

Type IV pili is an important virulence factor for most Gramnegative bacilli due to its adhesion, and is a prerequisite for initial bacterial adhesion to host cells.When Burkholderia melioides reaches the surface of the host cell membrane, its type IV pili contract and extend through filament polymerization and depolymerization[17], move along the surface and come into close contact with host cells, binding with host epithelial cells to perform adhesion[18].As a result, Burkholderia melioides can infect multiple organs throughout the body.When PliA, a special structural protein of type Ⅳ pili, is absent, the adhesion and damage of bacteria to host cells are significantly reduced.

2.Virulence factors of Burkholderia pseudomallei

2.1 BimA

The intracellular movement of Burkholderia melioides in host cells is mediated by host actin.Intracellular motility associated protein(BimA) is an actin secreted by bacteria.After adhering to host cells,Burkholderia melioides secretes BimA, which induces host actin aggregation and enables the bacteria to cross the cell membrane and reach into the cell[19].Induced actin filaments allow Burkholderia melioidae to move within the cell, while forming actin based cell membrane protrusion structures that allow it to move from cell to cell and infect other cells, contributing to multiple nuclei

The formation of multi-nucleatedgiantcells (MNGCS) increases the living space in the cell, and can escape phagocytosis of immune cells and spread throughout the body[20, 21].At the same time,BimA has been shown to be associated with the development of pneumonia, and mutations in the BimA gene, known as BimABm mutations, have been associated with the development of melioid encephalomyelitis.Such mutations have not been found in Asian strains, where the incidence of encephalomyelitis is low.

2.2 Type III secretion system

Type III secretion system (T3SS), is a complex structure composed of a variety of protein molecules, and Burkholderia pseudomallei is known to have three sets of T3SS, of which only t3ss-3 is the main active component[22].The main role of T3SS is that, after bacterial adhesion to the host cell, it can induce host actin rearrangements that allow it to rapidly escape into the cytoplasm, dominating the phagocytic escape of B.melioides in the host cell.T3SS can secrete a specific effector protein bopc, and experiments by Gong l et al demonstrated that inactivation of bopc leads to reduced bacterial survival in the phagosome[23,24].

2.3 Type VI secretion system

Type VI secretion system (T6SS), a bacterial secretion system widely present in gram negative bacteria[25].The t6ss in B.melioides was named tss-5[26].The t6ss is a multigene encoded multimolecular complex composed of a membrane complex, a substrate complex,and a phage like structure.The valine glycine heavy protein G(vgrg), hemolysin regulatory protein (HCP) in phage like structures plays a key role[27].When the t6ss is activated, the vgrg in the phage, can perforate the cell membrane, and release multiple effector proteins; The virulence of effector proteins and other small molecules, which directly enter target cells via HCP, can lead to cell membrane vacuolization, inhibit cell growth, and disrupt bacterial cell morphology[28].

T6SS also plays a key role in promoting the formation of MNGC.Studies have found that when Burkholderia melioides loses Hcp and VgrG, MNGC cannot be formed and cell fusion cannot occur[29, 30].Lack of T6SS, resulting in reduced transmission of bacteria between cells, is an important virulence factor of Burkholderia melioides.

2.4 Capsular Polysaccharide (CPS)

When the growth of Burkholderia melioides is observed under a microscope, a layer of radioactive filaments can be seen on the outside of the bacteria, which is the polysaccharide of the bacteria capsule.Studies have shown that the polysaccharide of the capsule can protect it from being phagocytosis, and the main reason is that the polysaccharide of the capsule has anti-lysosome effect[31].Burkholderia melioides is known to be capable of latent infection for several months or even years, which may be the mechanism leading to latent infection of melioids[32].

2.5 Burkholderia lethal factor 1 (BLF1)

Burkholderia lethal factor 1 (BLF1), the first discovered lethal factor of the genus Burkholderia, causes host cells to lose the helicase activity of mRNAs and inhibits the translation initiation stage of proteins, and the synthesis of proteins is inhibited and cells damaged to lose their activity[32,33].

3.Drug resistance mechanism

Burkholderia meliformis has one of the largest bacterial genomes(7.2Mb) and contains a large number of virulence determinants[34], which makes its clinical manifestations diverse and lack of specificity, making it difficult to study the mechanism of drug resistance.It is now known that Burkholderia melioidis has a natural drug resistance mechanism, which is resistant to many common antibiotics in clinical practice, such as quinolones, firstgeneration and second-generation cephalosporin, penicillin, etc.Under experimental conditions, it can be seen that it is sensitive to ceftazidime, carbapenems and other antibiotics.With the extension of diagnosis and treatment time, improper selection of antibiotics can lead to the increase of resistance of Burkholderia melioides to tetracycline and other antibiotics.Among them, ceftazidime is the preferred drug for Burkholderia melioides[35], and early first-line use can significantly reduce clinical symptoms of patients, which can be used as a key point to distinguish pneumonia caused by Klebsiella pneumoniae with high virulence.At present, the resistance mechanism of Burkholderia melioidosis has not been clarified,which may be related to the changes of efflux pump and antibiotic target sites[36, 37].

3.1 Efflux pumps

Efflux action of bacteria is an important mechanism for the development of drug resistance, and efflux systems are currently classified into six superfamilies, of which three efflux pumps amrab OPRA, bpeab oprb, and bpeef OPRC, all of which are RND(drug resistance nodulating family of cell differentiation)-type efflux pumps, are present on the cell membrane of Burkholderia pseudomallei[38, 39].Efflux pumps confer resistance to some type of antibiotic in B.melioidosis by altering its own regulators, Amra and amrb genes are important constituent genes of amrab OPRA, which enables the efflux of aminoglycosides as well as macrolides[40].The rest of the efflux pump resistance mechanisms have not been studied clearly.

3.2 Antibiotic target site changes and others

In a study by Muhammad Shafiq et al., 8 clinical isolates of Burkholderia melanoidae were obtained from Guangdong Province in 2018-2019, and sequenced by Illumina NovaSeq platform, multisite sequence typing (MLST) and single nucleotide polymorphism(SNP) analysis.The diversity and epidemiological characteristics of the strains were analyzed.It was concluded that the resistance of Burkholderia melioides to ceftazidime may be caused by the mutation of PenA(the gene encoding highly conserved betalactamase A).The outer membrane protein Omp38, a pore protein on the outer membrane of melioidosis, is involved in encoding the outer membrane pore of melioidosis.The structure of the outer membrane pore reduces the permeability of melioidosis to β-lactam.gyrA gene was found in all strains, and Viktorov et al.confirmed that gyrA mutation was the cause of quinolone resistance[41].

4.Discussion

The above are the pathogenic and drug-resistant mechanisms of Burkholderia pseudomallei, the mode of chronic latent and acute onset of melioidosis, and the relationship between hosts remain to be verified.Current relevant studies revealed that the long-term survival of B.melioidosis in host cells may be related to its ability to cause host cell autophagy.After experimentally infecting Burkholderia melioides into THP-1 cells, the damaged mitochondria of the host cells were observed after 2 h of infection, and it was observed that the GFP-LC3 colocalization with autophagosomes was significantly increased in the mitochondria of the host cells after infection (P＜ 0.01), suggesting that the occurrence of mitophagy in the host cells was induced after B.melioidosis infection[42].How to prevent the above mechanisms from occurring, remains to be further investigated.

Since Burkholderia melioides was first discovered, the mechanism of infection and drug resistance has not been clearly defined.With the convenience of transportation, the incidence in non-epidemic areas is gradually increasing, and due to the lack of knowledge about the bacteria, the misdiagnosis rate increases[2].For the structure of T6SS, the detection of hemolysin regulatory protein (Hcp) specific IgG antibody is an important early screening index at present[43].Although HCP1-specific IgG antibody detection is an important target, false negatives are prone to occur in people with early infection, severe infection and low immunity[44].Second-and thirdgeneration bacterial gene sequencing methods (NGS) have improved the diagnosis rate of Burkholderia melioidosis[45].The positive rate of alveolar lavage fluid was high in clinical specimens.

With this review, the current pathogenic and drug-resistance mechanisms of Burkholderia pseudomallei are discussed.Future perspectives on the diagnosis and treatment of Burkholderia pseudomallei: (1) using sequencing methods such as ngs to make a rapid and definite diagnosis.Metagene next-generation sequencing(mngs), which rapidly and unambiguously infects pathogens at an early stage, allows drug resistance detection[46].(2) A metagenomics approach is adopted to more fully mine the novel genomics of bacteria for further antimicrobial adjuvant development.(3) Whether targeting the T3SS, T6SS, Bima, and other mechanisms that could lead to immune escape against B.meliloti will allow further studies of interference and vaccines.(4) The lysozyme action of the capsular polysaccharide may be the mechanism responsible for the latent infection of Burkholderia pseudomallei,and further studies with molecular probes against this structure are warranted,long before the primary screening for the disease.(5) Clinically visible cases of melioidosis with iron overload, which appear to be associated with an increased incidence of Burkholderia melioides, may indicate that iron plays a more important role in the pathogenesis than is known and may be further investigated in relation to this.