Development of Monoclonal Antibodies Specific to Burkholderia Pseudomallei for Diagnosis of Melioidosis

University of Nevada, Reno

Development of monoclonal antibodies specific to Burkholderia pseudomallei for diagnosis of melioidosis

A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science in Cell and Molecular Biology

Farida D. Handayani

Dr. David AuCoin/Thesis Advisor

August 2010

THE GRADUATE SCHOOL

We recommend that the thesis prepared under our supervision by

FARIDA DWI HANDAYANI

entitled

Development of monoclonal antibodies specific to Burkholderia pseudomallei for diagnosis of melioidosis

be accepted in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

Dr. David Aucoin, Advisor

Dr. Gregory Pari, Committee Member

Dr. Thomas Kozel, Committee Member

Dr. Normand Leblanc, Graduate School Representative

Marsha H. Read, Ph. D., Associate Dean, Graduate School

August, 2010

Abstract

Burkholderia pseudomallei, the causative agent of melioidosis, is a Gram- negative intracellular bacterium. This pathogen is a major contributor to community- acquired septicemia in Southeast Asia and Northern Australia, and notoriously difficult to diagnose. At present, a goal of our laboratory is to develop an immunoassay capable of accurately detecting shed or secreted bacterial antigens in patient body fluids. Here, we report on a group monoclonal antibodies (mAbs) that have been generated against B. pseudomallei for this purpose. mAbs 3C5 and 4C7 were found to react with the capsular polysaccharide (CPS) and lipopolysaccharide (LPS), respectively. This was confirmed by the inability of 3C5 to react with a B. pseudomallei CPS-deletion mutant, and its insensitivity to proteinase K treatment. mAb 4C7 was reactive with a 25-75 kD polysaccharide in a ladder pattern, a characteristic of LPS binding. mAb F5C3 is reactive with the B. pseudomallei flagellin protein monomer at 43 kD, as was confirmed by mass spectrometry of the reactive protein. We determined the specificity of mAbs 3C5, 4C7 and F5C3 with lysate from 12 various Burkholderia species and other common pathogenic bacteria. mAb 3C5 was reactive with B. mallei, but not B. thailandensis (both of these bacteria are closely related to B. pseudomallei) and mAb 4C7 was reactive with both B. mallei and B. thailandensis. mAb F5C3 was not reactive with B. mallei but was reactive with B. thailandensis although, at a slightly lower molecular weight (41 kD). All of the mAbs were tested for their ability to detect antigen in serum and urine samples from melioidosis patients. Antigen was not detected in serum, however, mAbs 3C5 and

F5C3 were determined to be superior to mAb 4C7 in their ability to detect antigen in urine. ii

DEDICATION

THIS THESIS IS DEDICATED TO MY SON AND MY HUSBAND, WHO ALWAYS INSPIRE ME TO BE A BETTER PERSON, AND ALSO FOR MY MOM AND DAD, MY FIRST EDUCATORS WHO CONTINUOUSLY TEACH ME WITH THEIR LOVE.

iii

Acknowledgements

I wish to express my profound gratitude and indebtedness to my advisor Dr. David AuCoin for his constant encouragement and guidance in my study and research, and for allowing me to be part of his lab, but above all I would like to thank him for helping me to develop critical thinking skills and become a competitive scientist.

In addition, I would like to thank Dr. Greg Pari, Dr. Thomas M. Kozel and Dr. Normand Leblanc as my thesis committee for their support in assisting me in obtaining my master's degree.

I gratefully acknowledge Staff Research Associates: Dana N. Nuti, MS, Reva B. Crump, MS, and Peter Thorkildson from our laboratory in the Department of Microbiology and Immunology for their patience, assistance and guidance during the course of my research.

I would also like to thank my research colleagues in our laboratory at the University of Nevada Reno for their stimulating discussions and for maintaining a cheerful working atmosphere. Thank you to all my friends who supported me during my program; especially Sindy Chaves, Susan Hoover, Bahay Gulle, Serife Ozger, Ni Ketut, Manila Hada, Emanuelle Kuhn, without a doubt I cannot repay our true friendship.

Last but certainly not least, I would like to extend my everlasting gratitude to my beloved son Rayyan Ilham Wahyudi, my husband, parents, and all my family for their sacrifices, perpetual support, patience and understanding.

Table of Contents

Title Page Page i Abstract Page ii Dedication Page iii Acknowledgements Page iv Table of Contens Page v List of Tables Page vi List of Figures Page vii

I. Introduction Page 1 Burkholderia pseudomallei Page 1 Melioidosis Page 3 Laboratory diagnosis Page 5 Capsular polysaccharide Page 8 Lipopolysaccharide Page 9 Flagellin Page 11 II. Specific Aim Page 12 III. Material and Methods Page 13 Monoclonal antibodies and bacterial strains Page 13 Gel electrophoresis and western blot analysis Page 13 ELISA methods Page 14 Immunoflourescense assay Page 15 IV. Results Page 16 Characterization of mAb 3C5 Page 16 Characterization of mAb 4C7 Page 21 Characterization of mAb F5C3 Page 26 V. Discussions Page 33 Characterization of mAb 3C5 Page 34 Characterization of mAb 4C7 Page 36 Characterization of mAb F5C3 Page 38 VI. Conclusion Page 40 VII. References Page 41

List of Tables

Table 1. mAbs used in the study Page 16 Table 2. Mass spectrometry analysis identifying the corresponding spots Page 28 Table 3. The immunoreactivity of mAbs 3C5, 4C7, and F5C3 Page 32

Table of Contents

Figure 1. Western blot analysis of proteinase K treated cells with mAb 3C5 Page 17 Figure 2. Western blot analysis of B. pseudomallei CPS mutant with mAb 3C5 Page 17 Figure 3.Western blot analysis of mAb 3C5 with B. pseudomallei, Page 18 B. thailandensis, B. mallei Figure 4. Antigen capture ELISA for mAb 3C5 Page 19 Figure 5. IFA of mAb 3C5 to B. pseudomallei. Page 20 Figure 6. IFA of mAb 3C5 to with Burkholderia species Page 20 Figure 7. Detection of CPS in melioidosis patients urine with mAb 3C5 Page 21 Figure 8. Western blot analysis of proteinase K treated cells with mAb 4C7 Page 22 Figure 9. Western blot analysis of B. pseudomallei CPS mutant with mAb 4C7 Page 22 Figure 10. Western blot analysis of specificity of mAb 4C7 Page 23 Figure 11. Analysis of mAb 4C7 binding to Burkholderia species. Page 24 Figure 12. Antigen-capture ELISA of B. pseudomallei extract to mAbs 4C7 Page 25 Figure 13. Immunoflourescence assay mAb 4C7 binds to B. pseudomallei. Page 26 Figure 14. 2D Western blot analysis for identifying B. pseudomallei flagellin Page 27 Figure 15. The specificity of mAbs F5C3 by Western blot Page 29 Figure 16. Checkerboard ELISA of mAb F5C3 to rfliC Page 30 Figure 17. Flagellin detection in urine samples from melioidosis patients Page 31

Introduction

Burkholderia pseudomallei

Burkholderia pseudomallei is the causative agent of melioidosis, an emerging disease endemic to Southeast Asia and northern Australia (Cheng and Currie, 2005). This

Gram-negative bacterium has the ability to survive in soil and water and is commonly found in rice paddy fields in Thailand as well a southern and central Vietnam (Parry at al., 1999). Studies have shown that 20% of cases of septicemia and 40% of cases of sepsis related mortality that occurred in Thailand are principally due to B. pseudomallei

(White, 2003; Wiersinga, W.J., et.al., 2006). In northern Australia, the mortality rate is reported still high at 20% (White, 2003).

The Burkholderia genus consists of more than 40 species (Vandamme, et.al.,

2007). Prior to 1992, Burkholderia pseudomallei was identified as Bacillus pseudomallei,

Bacillus whitmorii, Malleomyces pseudomallei, and as Pseudomonas pseudomallei

(Cheng and Currie, 2005; Dance, 1991). A number of these species cause serious human disease. The most pathogenic members are B. pseudomallei, B. mallei and B. cepacia. B. mallei is the causative agent of glanders, a disease commonly found in horses, whereas B. cepacia complex are known as opportunistic pathogens that cause life-threatening infections in cystic fibrosis patients (Vandame, 2007). B. pseudomallei and B. mallei have been classified as a category B select agent by the US Centers for Disease Control and Prevention (Holden, M.T. et.al., 2004). This is primarily due to their ability to be aerosolized along with causing infections with relatively low doses.

The B. pseudomallei genome consists of two chromosomes, which are 4.07 Mb and 3.17 Mb long (Holden, M.T., et.al., 2004). Most of the large chromosome consists of genes associated with cell growth and metabolism, whereas the smaller chromosome encodes genes that play a role in adaptation and survival ability in diverse environments.

Genomic islands make up a small portion of the B. pseudomallei genome (6%), which are assumed to have been acquired through horizontal gene transfer (Nierman, W.C., et.al.,

2004). These genomic islands are absent in B. thailandensis genome as well as in the B. mallei genome (Nierman, W.C., et.al., 2004). Experts still debate the role of these genomic islands and whether those parts are involved in pathogenesis. In 2008, Tuanyok and colleagues studied the diversity of genomic islands in five different strains of B. pseudomallei and hypothesized that strain to strain variation in genomic islands may be one of the major reasons for the diverse range of clinical manifestations among melioidosis patients (Tuanyok, et.al., 2008).

B. pseudomallei is an intracellular bacteria which can invade epithelial cells, survive and proliferate inside phagocytes, and escape from endocytic vesicles

(Pruksachartvuthi, S., 1990). B. pseudomallei has the ability to resist host antimicrobial peptides, such as defensins, protamine, and iNOS-inducible Nitric-oxide synthase, which is known to have an important role in the killing of intracellular bacteria

(Pruksachartvuthi, S., 1990). In addition, B. pseudomallei can induce apoptosis of both phagocytic and non-phagocytic cells. Once the bacteria successfully infect a cell, they will form actin-based membrane protrusions and can spread cell to cell and then induce the formation of multinucleated giant cells by cell fusion (Kespichayawattana, et.al, 3

2000). This feature is an exception among bacterial intracellular pathogens (Wiersinga,

W.J., et.al., 2006).

B. pseudomallei infection may occur through skin abrasions, aspiration of contaminated water, inhalation, and possibly ingestion (Cheng and Currie, 2005). B. pseudomallei can infect a wide range of animals typically terrestrial and aquatic mammals, including livestock, and cause economic losses (Dance, 1991). B. pseudomallei transmission from animal to human has rarely occured, although person-to- person transmission and laboratory-acquired infections have been documented (Dance,

1991; Cheng and Currie, 2005). The ability of B. pseudomallei to survive in soil and water environments is quite high (Inglis, T.J. et. al. 2000). It has been reported that B. pseudomallei can survive for years in distilled water and can also enter and survive within free-living amoebae belonging to the genus Acanthamoeba (Inglis, T.J. et. al.

2000). Acanthamoeba is one of the common protozoa frequently found in soil and in fresh water. In one outbreak reported in Western Australia, they found coexistence between B. pseudomallei and Acanthamoeba (Inglis, T.J. et. al. 2000). Several

Acanthamoeba species were recovered from water specimens collected during the early outbreak and in further environmental investigations over the following year, prompting a study into possible interactions between Acanthamoeba species and B. pseudomallei

(Inglis, T.J. et. al. 2000).

Melioidosis

Alfred Whitmore first described melioidosis as a “glanders-like disease”

(Whitmore’s disease). He differentiated it from glanders, which is caused by B. mallei, by clinical and microbiological features (Cheng and Currie, 2005). Melioidosis came from 4 the Greek words; “melis” (distemper of asses) and “eidos” (resemblance). The disease is endemic to Thailand and Northern Australia, and is sporadic in China, Korea, the

Philippines, India, Indonesia and West Africa (Cheng and Currie, 2005; Dance et.al,

1999). Some cases have been reported in Europe and North America due to traveling activities to the endemic areas (Cheng and Currie, 2005).

Melioidosis cases in tropical areas tend to increase during the rainy season. This is believed to occur by aerosol transmission of the bacteria, which increases the cases of pulmonary infection. A report showed that melioidosis in pneumonia patients in Northern

Australia was caused by heavy rainfall and high winds (Weirsinga, et.al., 2006). Another risk factor for melioidosis came about after the 2004 tsunami in Southeast Asia. An epidemiological study found 10 cases of melioidosis in Banda Aceh, Indonesia, with patients having pneumonia-like symptoms (Peacock, 2006). In addition this report also found that some surviving residents from the tsunami in Thailand were later found to test positive for melioidosis.

B. pseudomallei can cause a wide spectrum of clinical diseases. For this reason, melioidosis is sometimes called “the great imitator.” or called “the great mimicker” due to the variation of clinical signs and symptoms after infection (Peacock, 2006). Patients who are more susceptible to develop a melioidosis infection are those who are immuno- compromissed due to diabetes mellitus, renal disease, alcoholism and thalassemia because of an association with impairment of neutrophil function (Brent, et.al., 2007).

The incubation period of melioidosis is nine to ten days. However, many infections are initially subclinical but may result in latency and delayed manifestation, even after several decades. Due to the latency manifestation, melioidosis is identified as a biological 5 time bomb (Ngauy et.al, 2005) or “sleeping with the enemy” (Gan, 2005). This is due to the fact that B. pseudomallei can be latent for many years and soldiers returning from

Vietnam may develop the disease years later. Re-infection of B. pseudomallei does not give any protection for the susceptible individuals to get another infection (Wiersinga et.al., 2006). Clinical signs and symptoms include septicemia, pneumonia, bone and soft tissue infections, abscesses, mycotic aneurysms, lymphadenitis, and parotitis. Some cases have been mistakenly identified due to similarity of the disease symptoms, as a flu-like syndrome (Ngauy et.al, 2005).

Laboratory diagnosis

B. pseudomallei infection is difficult to diagnose and may lead to misdiagnosis due to lack of understanding of the disease and inappropriate identification methods.

Speed of diagnosis is important especially in developing countries where cases of acute- septicemia may be treated with antibiotics that are ineffective against B. pseudomallei

(Peacock, 2006). The bacteria is resistant to many antibiotics including penicillin, cephalosporins, macrolides, rifamycins, colistin and aniboglycosides, but is susceptible to amoxicillin, ceftazidime and carbapenems (White, 2003). Confirmation of the disease depends on bacterial isolation from clinical specimens, a technique that is difficult to practice in some melioidosis endemic areas and is time consuming. Melioidosis cases represent “the tip of the iceberg”, which means the true number of cases is probably much higher than reported due to difficulties in diagnosis (Wiersinga et.al., 2006; Dance,

1991).

Isolation of B. pseudomallei from cultures of patient samples remains the “gold standard” for the diagnosis of melioidosis. Ashdown’s selective agar is commonly used 6 for the growth of B. pseudomallei because the medium contains several selective agents, which suppress the growth of other bacteria (Peacock, 2005). Often times culturing is challenging because the levels of bacteremia is extremely low (<0.1 cfu/ml in blood)

(Wuthiekanun, 2007). Several biochemical kits such as API 20NE and RapID NF Plus have been used to identify B. pseudomallei infections. However, these tests have been shown to be inaccurate. In one report only 37% of B. pseudomallei isolates were correctly identified by API 20NE, and none were correctly identified by RapID NF Plus (Dance, et.al., 1989).

Many other different techniques have been used to diagnose melioidosis.

Polymerase chain reaction (PCR) can be used for the identification or detection of B. pseudomallei. Although the specificity for some PCR assays reach up to 100%, sensitivity remains low at 65%. This is most likely caused by low bacterial counts in blood that are common in B. pseudomallei infections (Gal, et al., 2005). Serological assays have been developed for the diagnosis of acute melioidosis. A rapid immunochromogenic cassette test (ICT) showed a lower specificity and sensitivity compared to the culture identification. The indirect haemagglutination assay (IHA) is commonly used to identify B. pseudomallei infection, IHA is a rapid and inexpensive assay; however, a large percentage of healthy individuals in endemic areas are seropositive. In one report, the ability of the IHA to diagnose a melioidosis infection in patients from northern Australia is quite low (Cheng, et.al., 2006).

Many immunological methods have been developed for the detection of B. pseudomallei antigen. Antigen detection is more reasonable and superior to antibody detection because it indicates active disease. This is suitable when used in the endemic 7 area of infection, where the background antibodies often interfere with the interpretation in the antibody assay (Sirisinha, et.al., 2000). B. pseudomallei antigens can be identified directly in tissues, wound exudates or body fluids by direct immunofluorescence or latex agglutination. Antigen tests including enzyme-linked immunosorbent assays (ELISAs) have also been developed for the exotoxin and other bacterial components; in addition, the antigen detection can be also identified by dot immunoassay or immunoblotting

(Western blotting) (Sirisinha, et.al., 2000). A study carried out by Steinmetz et.al. demonstrated that a mAb against exopolysachharide (EPS) specific to B. pseudomallei could be used as a rapid identification test for melioidosis infection by a latex agglutination assay (Steinmetz, et.al., 1999). Another study exhibited a rapid method to distinguish B. pseudomallei and B. thailandensis using monoclonal antibody-based latex agglutination test systems (Wuthiekanun, 2002). A competitive ELISA using a LPS mAb was also developed to recognize B. pseudomallei infections and this assay provided high specificity (Thepthai, et.al., 2005).

Accuracy and speed of diagnosis is very important, especially in rural areas where under diagnosis of melioidosis may exist due to the lack of laboratory equipment and lack of laboratory skills (Peacock, 2006). Monoclonal antibodies (mAbs) are known to be powerful tools, which can be used for antigen analysis, diagnostic and classification, as well as immunoprotection (Gal, et.al., 2005). Our laboratory has generated three mAbs that could potentially be used to develop an immunoassay for use in point-of-care settings. These mAbs are targeted to the capsular polysaccharide (CPS), lipopolysaccharide (LPS) and flagellin.

Capsular polysaccharide

B. pseudomallei produces an extracellular capsular polysaccharide with the structure -3)-2-O-acetyl-6-deoxy-β-D-manno-heptopyranose-(1, that was previously described as O-PS I (Reckseidler et.al., 2001). It is considered as capsular polysaccharide based on its high molecular weight and genetic homology with other organisms

(Weirsinga, 2006).

In experimental animal models it has been shown that the capsular polysaccharide is required for B. pseudomallei virulence and involves resistance to phagocytosis

(Nelson, et.al., 2004). It has been suggested that CPS of B. pseudomallei is important in environmental protection, immune system evasion and attachment to epithelial cells

(Nelson, et.al., 2004 and Puthucheary, 1996).

Microarray analysis showed that CPS was up-regulated significantly after Syrian

Golden hamsters were infected with B. pseudomallei and the capsule was shown by sequence analysis and immunoblot analysis to be present in B. mallei, but not B. thailandensis (DeShazer, et al., 2001). Reckseidler-Zenteno’s stated that there are four types of CPS, which are the CPS I, CPS II, CPS III and CPS IV. Their study revealed that

CPS I cluster is only present in B. pseudomallei and B. mallei, but not B. thailandensis

(Reckseidler-Zenteno, et.al., 2009). Several studies showed that CPS antibodies may be highly protective and can be used as a candidate vaccine. An IgG2b isotype of CPS antibody was reported to have provided increased protection to mice (Nelson, et al.,

2004).

Lipopolysaccharide

Lipopolysaccharides (LPSs) are commonly found on the surface of Gram- negative bacteria. Bacterial LPSs consist of three components, lipid A, the core polysaccharide and the O-Antigen. Lipid A anchors LPS to the outer leaflet of the bacterial membrane; the core polysaccharide consists of an oligosaccharide; and the O- antigen consists of long polysaccharide repeats. The O-antigen is the immunogenic potion of LPS and is the main reason for the different antigenic specificities among

Gram-negative bacteria (Schaechter, et.al., 1999).

The LPS of B. pseudomallei has been termed O-PS II and is unique and different from other Gram-negative organisms. The structure of the O-antigen has been determined to be a repeating disaccharide unit of -3)-β-D-manno-glucopyranose-(1-3)-6-deoxy-α-L- talopyranose-(1- (Perry, et.al., 1995). The LPS structures of B. pseudomallei seem identical with B. thailandensis (Brett, et.al., 1998). Most LPS of B. pseudomallei (96%) exhibits a typical ladder pattern that can be visualized by silver stain (Anuntagool, et.al.,

2006). The same study determined that other small percentage of the bacteria have different ladder patterns (3%), which is referred as an atypical ladder or B.pseudomallei

LPS without any ladder pattern (1%) but possessed a low molecular weight below the 29 kD. The LPS O-antigen structures of B. pseudomallei and B. mallei have been shown to be slightly different; B. mallei does not produce acetyl modifications at the O-4 position of L-6dTalp (Burtnick, et.al., 2002).

Host recognition of LPS is essential to initiate the immune response in Gram- negative bacteria through activation of Toll-like receptor (TLR) 4. LPS activates the cells of the immune system through a receptor complex that consists of a ligand-binding 10 molecule (CD 14) and TLR 4, which is the signal transducer (Wiersinga, et. al., 2006).

The lipid A portion of B. pseudomallei LPS has been shown to have longer fatty acids than the lipid A from enterobacteria; these longer fatty acids are predicted to decrease the recognition of B. pseudomallei LPS with CD14 on the macrophage cell surface, resulting in a reduced inflammatory response (Adler et.al., 2009).

It seems likely that antibody bound to surface expressed capsular polysaccharide

(CPS) and lypopolysaccharide (LPS) enhance phagocytosis by macrophages and neutrophils and may enhance subsequent killing of internalized bacteria (Jones, et.al.,

2002). The antibody specific for LPS O-PS from B. pseudomallei could be protective and this protection might be mediated by enhancement of phagocytosis leading to bacterial clearance (Jones, et.al., 2002)

B. pseudomallei LPS is a well established virulence factor (DeShazer et al. 1998).

One study determined that the level of antibody specific to B. pseudomallei LPS was significantly higher in melioidosis patients who survived than in those who died. These antibodies were also significantly higher in patients with nonsepticimic as opposed to septicemic melioidosis (Charuchchaimontri, et.al., 1999). This indicated that LPS is a potential component in immunological importance (Jones, et.al., 2002). An additional study determined a B. pseudomallei LPS mutant strain was attenuated in hamsters, guinea pigs and diabetic rats. This same LPS mutant was (i) more susceptible to complement dependent killing, (ii) showed increased internalization by macrophages and (iii) decreased intracellular survival (Adler et. al., 2009).

Flagellin

B. pseudomallei is a motile bacterium that produces two or four flagella

(DeShazer, et.al., 1997). The flagella are believed to be involved in B. pseudomallei pathogenesis (Chua et. al. 2003). B. pseudomallei is a saprophyte organism that obtains the nutrition from invasion of protozoa. The initial adhesion of B. pseudomallei to free- living protozoa Acanthamoeba astronyxis involves attachment via flagella (Inglis et.al.,

2000).

There are conflicting data on the involvement of flagella with virulence. A transposon mutant of the flagellin structural gene, fliC, was not attenuated in the diabetic rat or Syrian hamster melioidosis models (DeShazer, et.al., 1997). However, unlike wild type B. pseudomallei, the fliC mutant was not able to adhere to cells of the free living amoeba A. astronyxis, a crucial step to invade to the host (Inglis et.al., 2000). Another study confirmed that a B. pseudomallei fliC mutant was attenuated in BALB/c mice that were infected by either the intranasal or intraperitoneal routes (Chua, et.al., 2003). This mutant also showed a reduced number colony forming units in the lung and spleen of

BALB/c mice compared with the wild type (Chua, et.al., 2003).

Burkholderia genus consist of more than 40 species, but interestingly, not all of them has flagella. B. thailandensis has flagella (Chuaygud, et.al., 2008); in addition, B. mallei, an etiologic agent of glanders, is non-flagellated and non motile. Another species in Burkholderia genus, B. cepacia is a motile organism with polar flagella (Tomich, et.al., 2002).

Specific Aim

Melioidosis is an important health problem endemic in Southeast Asia and northern Australia. It has been reported that melioidosis has spread widely to other parts of the globe. Burkholderia pseudomallei is the causative agent of this disease and it is categorized as a potential bioterrorism agent. Melioidosis is difficult to diagnose and it has been known to be resistant to many antibiotics. Monoclonal antibodies are widely used as diagnostic tools and as a method to treat infections. The overall goal of this research is to construct an immunoassay that could potentially be used as a rapid method to detect antigens in melioidosis patients’ urine or blood. In order to produce effective immunoassays, the specificity and characterization of the mAbs must be determined.

Immunoassays for antigen detection have a number of benefits to identify infections.

First, they are a proven technology; secondly, they avoid false positive results due to antibiotic treatments; they can detect infection at a distant site; provide rapid and low cost diagnostics, and finally, they can be performed readily by any laboratory personnel in rural endemic areas with minimum equipments. Therefore, in this study we would like to investigate the specificity of B. pseudomallei mAbs 3C5, 4C7 and F5C3, as well as to determine whether those mAbs could be used to detect antigen in urine of melioidosis patients. We will investigate the cross reactivity of mAbs 3C5, 4C7 and F5C3 with 12 various Burkholderia species and other common pathogenic bacteria.

Material and Methods

Monoclonal antibodies and bacterial strains. A panel of monoclonal antibodies

(mAbs) was previously produced from Balb/c mice immunized with heat-inactivated B. pseudomallei that was activated at 80°C for 2 hours. Three of these antibodies, 3C5, 4C7, and F5C3, were selected for characterization (Table 1). Twelve Burkholderia species and seven other common microbial species have been used in this research to characterize those three mAbs. The bacterial strains used in this study are listed in Table 3. B. pseudomallei strain 1026B was obtained from Dr. Richard Bowen (Colorado State

University). B. thailandensis and B. mallei were bought from ATTC, and the irradiated B. mallei was acquired from the Critical Reagents Program Antigen Repository (BEI

Resources, VA, USA). Additionally, the B. cepacia group was obtained from the

Burkholderia cepacia Research Laboratory and Respiratory at the University of

Michigan, USA.

Gel electrophoresis and Western blot analysis. Sodium Dodecyl Sulfate

Polyacrylamide Gel Electrophoresis (SDS-PAGE) was performed with a 10% ready

(precast) gel from Biorad. Samples were mixed with Laemmli 2x concentrate buffer

(Sigma) 1:1; which contains 4% SDS, 20% Glycerol, 10% 2-mercaptoethanol, 0.004% bromphenol blue and 0.125 M Tris HCL, pH proximally 6.8, and boiled for 10 minutes.

If required, proteinase K (1 volume; 3.3 mg in 1 ml Laemmli sample buffer) was added after the boiling section and incubated for 1 hour at 60ºC. Samples were loaded into the gel then separated at 150 V for 50 minutes with 1x Tris-Glycine-SDS Running buffer 14

(Boston BioProducts cat# BP-150). The gels were transferred onto Immun-Blot PVDF membrane following with either tank blotting or semidry blotting. Subsequently, the proteins in the gel were either stained with Coomassie Brilliant Blue or electrotransferred onto polyvinylidene fluoride (PVDF) membrane. The PVDF membranes were then blocked using TBST 5% milk for at least 15 minutes to overnight then following probed using purified mAbs diluted to 1: 10,000 in TBST 5% milk and incubated for an hour.

The membrane was then washed 2x in TBST and incubated for 30 minutes with the secondary antibody (Horseradish-peroxidase-conjugated goat anti-mouse IgG from

Southern Biotech cat# 1030-05) in 1:10,000 dilution. Immunoreactive bands were developed with a substrate solution chemiluminescent (Pierce cat# 34077). A silver staining kit was used to identify carbohydrate components in the cells (Pierce cat#

24612).

ELISA methods. An enzyme-linked immunosorbent assay (ELISA) was used to characterize mAbs 3C5 and 4C7 by antigen capture; and mAb F5C3 by checkerboard

ELISA. Briefly, flat-bottomed Immulon IB 96–wells microtiter plates (Thermo cat#

3355) were coated with 1 µg/ml unlabeled 3C5 and 4C7 in PBS. The plates were incubated at room temperature overnight. The next day, the plates were washed in PBS and incubated for 2 hours in PBST milk (PBST containing milk powder 1% w/v) followed by a washing step with PBST (PBS containing Tween-20 0.05% v/v). B. pseudomallei crude extract was prepared in PBST milk with a starting concentration of

1000 ng/ml for mAb 3C5 and 8000 ng/ml for mAb 4C7. The antigen was serially diluted and the plates were incubated for 1.5 hours at room temperature. The plates were washed 15 with PBST milk and horseradish peroxidase (HRP) conjugated mAb 3C5 or 4C7 was added at 1:10,000 dilution and incubated for an hour at room temperature. After the final wash in PBST, 100 µl tetramethylbenzidine (TMB) peroxidase substrate (Kirkergaard &

Perry Laboratories; cat# 50-76-00) was added to each well for 30 minutes. The reaction was stopped by adding phosphoric acid and the signal immediately read at OD450.

Immunofluorescence assay. 1 x 108 cfu suspension of whole killed bacteria in PBS was smeared onto a microscope slide and allowed to dry. Cells were fixed with methanol and repeatedly the slides were washed in water. The slides were then incubated for 1 hour in

1% BSA (Invitrogen cat# P2046) as a blocking step to reduce non-specific binding. The slides were further washed in water before incubated with primary mAbs in BSA at a concentration of 200 µg/ml for 1,5 hours at room temperature. After washing by dipping in water, prepared Alexa Fluor 555-labelled goat anti-mouse IgG (Invitrogen cat#

A21424) was diluted 1: 2000 in BSA and incubated for 1 hour at room temperature. The slide was then rinsed for the final washing, and allowed to dry completely before being mounted with Vectashield (Vector Laboratories). The slides were observed under an epifluorescence microscope (Nikon Eclipse E800) with a built-in confocal microscope

(Nikon C1 from Nikon instruments).

Results

The overall objective of this study was to generate a variety of mAbs that could be used to accurately detect the presence of the infectious bacterium B. pseudomallei in patient urine in the form of a non-invasive, point of care diagnostic. Previously, our laboratory has generated a group of mAbs reactive with various membrane components of B. pseudomallei. Three mAbs termed 3C5, 4C7, and F5C3 were among this group and were selected for further characterization. Additional investigation showed that the mAbs were able to bind three distinct virulence factors of B. pseudomallei namely the capsular polysaccharide, lipopolysaccharide, and flagellin respectively.

Table 1. mAbs used in this study

mAb Isotype Target antigen

3C5 IgG3 Capsular polysaccharide

4C7 IgG3 Lipopolysaccharide

F5C3 IgG1 Flagellin

Characterization of mAb 3C5

Western blot analysis showed that mAb 3C5 binds to a high molecular weight band of B. pseudomallei lysate at an estimated 250 kD. Treatment of B. pseudomallei whole cell lysate with proteinase-K did not change the binding ability of 3C5 to the high molecular weight band; indicating that mAb 3C5 is reactive with a polysaccharide or carbohydrate of B. pseudomallei (Fig. 1).

Figure 1. Western blot probing B. pseudomallei strain 1026b whole cell lysates with mAb 3C5. mAb 3C5 binding to untreated (lane 1) and proteinase K treated (lane 2) whole cell lysates.

To further investigate the binding characteristics of mAb 3C5, we probed a capsule deleted mutant (SRM117) of B. pseudomallei with mAb. This mutant was created from parental strain 1026b by transposon mutagenesis (Reckseidler-Zenteno, 2005;

DeShazer, 1997). Additionally, mAb 3C5 was not reactive with this mutant (Fig. 2); indicating that mAb 3C5 reacts specifically with CPS. Taken together, these results show that mAb 3C5 is specific to the capsular polysaccharide (CPS) of B. pseudomallei.

Figure 2. Western blot analysis of a B. pseudomallei CPS mutant. mAb 3C5 is unreactive to B. pseudomallei CPS mutant (lane 1), but reactive to the wild type (lane 2). 18

The specificity of mAbs must be determined if they are to be used in the construction of an antigen capture immunoassay. Therefore, we needed to determine if mAb 3C5 bound to other members of the Burkholderia genus as well as other common pathogenic bacteria. By Western blot analysis, we determined that mAb 3C5 is not reactive with B. thailandensis but is reactive with B. mallei (Fig. 3). In addition we found that mAb 3C5 is not reactive with nine species of B. cepacia group and other common pathogenic bacteria, such as Pseudomonas aeruginosa, Streptococcus pneumoniae, Klebsiella pneumoniae, and Staphylococcus aureus (data is not shown).

Figure 3. Western blot analysis of mAbs 3C5 with B. pseudomallei (lane 1), B. thailandensis (lane 2) and B. mallei (lane 3). All samples received proteinase K treatment. mAb 3C5 is reactive with B. pseudomallei and B. mallei; however, this mAb is not reactive with B. thailandensis.

Then, we examined the binding characteristics of mAb 3C5 to crude B. pseudomallei extract using an antigen capture ELISA. We used 1 µg/ml of mAb 3C5 in the solid phase following two fold serial dilutions of crude B. pseudomallei extract (Fig.

4). The sample used was a crude extract of B. pseudomallei in which all that was known 19 was the total protein concentration. Therefore, we could not determine the sensitivity of the mAb.

Figure 4. Antigen capture ELISA was constructed with mAb 3C5 in the solid phase (1 µg/ml). Crude B. pseudomallei extract was serially diluted in the fluid phase followed by detection with HRP-labeled mAb 3C5 (1µg/ml). The starting dilution of the crude B. pseudomallei extract was 1000 ng/ml. mAb 3C5 could detect the B. pseudomallei extract at a starting concentration of 1µg/ml crude extract.

Immunofluorescence assay (IFA) was performed to visualize the capsular reaction of mAb 3C5 with whole-cell, heat-killed B. pseudomallei. Cells were plated on a microscope slide that was then incubated in 200 µg/ml of mAb 3C5, followed by labeling with anti-mouse Alexa Fluor 555 (1: 2000). mAb 3C5 reacted with B. pseudomallei on the surface of the bacterial cells (Fig. 5). Other Burkholderia species were also investigated in the IFA with mAb 3C5, such as B. thailandensis, B. mallei and B. cepacia

(Fig. 6).

Figure 5. Immunofluorescence assay of the binding pattern of mAb 3C5 to B. pseudomallei. Differential interference contrast (DIC) microscopy of B. pseudomallei (left), the center picture shows binding of mAb 3C5 to capsular polysaccharide of B. pseudomallei using Alexa Fluor 555 as secondary antibody. Merged images between the DIC and the fluorescence figure (right panel) which shows B. pseudomallei capsule surrounded by mAb 3C5.

B. pseudomallei B. thailandensis B. mallei B. cepacia

Figure 6. IFA with B. pseudomallei CPS mAbs. First, differential interference contrast images were taken followed by fluorescence images of mAbs 3C5. Alexa Fluor 555- labelled secondary antibody was used in all IFA experiments.

To further investigate the use of mAb 3C5 as a rapid diagnostic of melioidosis, the mAb 3C5 was used to detect antigen in the urine patient from Thailand by western blot analysis and ELISA (Fig. 7). Five patients’ urine samples that have been proved positive for B. pseudomallei were tested. All of the samples were passed through a 0.22

µm filter; therefore, no whole cells were present in the samples.

A B

Figure 7. Detection of CPS in melioidosis patients urine with mAb 3C5. (A) Western blot with patient urine probed with mAb 3C5 at 1:5000 dilution (1mg/ml stock). (B) Antigen capture ELISA with unlabeled mAb 3C5 in the solid phase (1 mg/ml). 100 µl of patient urine was serially diluted across a 96-well plate. HRP-labeled mAb 3C5 was used in the indicator phase.

Characterization of mAb 4C7

Commonly, the lipopolysaccharide (LPS) of Gram-negative bacteria has a specific ladder pattern by western blot due to O-antigen repeats. mAb 4C7 was found to bind its target antigen in a similar pattern suggesting that it may be specific to LPS.

Western blot analysis also showed that mAb 4C7 is insensitive to proteinase-K treated cells further indicating its interaction with LPS (Fig. 8). 22

Figure 8. Western blot probing B. pseudomallei (strain 1026b) whole cell lysates with mAb 4C7. mAb 4C7 binding to untreated (lane 1) and proteinase K treated (lane 2) whole cell lysates.

In addition, when we probed B. pseudomallei capsule delete mutant cells with mAb 4C7, we found that there was still LPS detected. We proposed that B. pseudomallei capsule deleted mutant still contains LPS (Fig. 9).

Figure 9. Western blot analysis of B. pseudomallei CPS mutant probed with mAb 4C7. mAb 4C7 is reactive with B. pseudomallei CPS mutant (lane 1). The wild type is shown on lane 2.

In order to verify the specificity of mAb 4C7 to B. pseudomallei, western blot analysis was performed using several members of the Burkholderia genus. The western 23 blot analysis provided evidence that mAb 4C7 binds to B. pseudomallei, B. mallei, and B. thailandensis (Fig. 10), but not to B. cepacia complex and other common pathogenic bacteria (data is not shown).

Interestingly, mAb 4C7 reacted stronger with B. mallei than with B. pseudomallei or B. thailandensis. Further studies demonstrated that mAb 4C7 appears to have increased reactivity to B. pseudomallei and B. thailandensis if B. mallei is not loaded on the same gel. These data suggest a possible variation in the quantity or quality of LPS produced by these pathogenic strains of Burkholderia. All pictures in Figure 10. were taken using the same exposure time.

Figure 10. Western blot analysis of mAb 4C7 with B. pseudomallei (lane 1), B. thailandensis (lane 2) and B. mallei (lane 3). All samples received proteinase K treatment. mAb 4C7 seems to react stronger with B. mallei than B. pseudomallei or B. thailandensis. However, mAb 4C7 appears to have increased reactivity to B. pseudomallei and B. thailandensis when B. mallei is not loaded on the same gel. All pictures in panel A and B were taken using the same time exposure.

This finding was further investigated by Coomassie staining. Subsequently, we ran two gels loaded with B. pseudomallei (1 µl), B. thailandensis (10 µl), B. mallei (1 µl), and B. 24 mallei (10 µl). One gel was stained with Coomassie Brilliant Blue and the other gel was electrotransferred onto PVDF membrane and probed using mAb 4C7. An interesting result was obtained; although only a small amount of B.mallei cells were detected by the

Coomassie stain, a better binding pattern was observed again between B. mallei to mAb

4C7 than to both B. pseudomallei and B. thailandensis (Fig. 10).

Figure 11. Analysis of mAb 4C7 binding to Burkholderia species. (A) Coomasie stained gel with 1 µl B. pseudomallei (lane 1), 10 µl B. thailandensis (lane 2), 1 µl B. mallei (lane 3), and 10 µl B. mallei (lane 4). (B) immunoblot profiles of the same Burkholderia species as well as identical concentrations as in panel (A) probing with mAb 4C7. Immunoblot demonstrated mAb 4C7 reacted stronger to B. mallei than B. pseudomallei and B. thailandensis, although only a small amount of B.mallei cells were detected.

Detection of LPS was also observed by antigen-capture ELISA for detection of

LPS (Fig. 12). 1 µg/ml mAb 4C7 was plated in the solid phase and incubated with a crude B. pseudomallei extract serially diluted in the fluid phase followed by detection with HRP-labeled mAb 4C7 (1 µg/ml). Figure 12 shows that mAb 4C7 was able to detect

LPS in the B. pseudomallei crude extract at a concentration of 250 ng/ml. However, it is not known how much of LPS was present in the B. pseudomallei extract. 25

Figure 12. Antigen-capture ELISA to investigate the binding of B. pseudomallei extract to mAbs 4C7. The starting dilution concentration of B.pseudomallei was 8000 ng/ml.

In addition to Western blots, immunofluorescence assays (IFA) were done with mAb 4C7. Binding of the mAb mirrored the results obtained with Western blots. As expected, mAb 4C7 reacted with B. pseudomallei, B. thailandensis and B. mallei on the exterior of the bacterial cells (Fig. 13). However, mAb 4C7 could not detect LPS in the melioidosis patient’s urine (Data not shown).

B. pseudomallei B. thailandensis B. mallei B. cepacia

Figure 13. Immunofluorescence assay mAb 4C7 binds to B. pseudomallei. Left panel is the differential interference contrast (DIC) microscopy of B. pseudomallei, the right panel shows binding of mAb 4C7 to outer layer of B. pseudomallei lipopolysaccharide.

Characterization of mAb F3C5

DeShazer et.al. have previously reported the molecular weight of the B. pseudomallei flagellin monomer to be 43 kD. mAb F5C3 was able to identify a 43 kD band B. pseudomallei protein by western blotting analysis, the reactivity of which was found to be proteinase K dependent. This finding led us to further investigate the specificity of this mAb. Mass spectrometry results confirmed that mAb F5C3 is reactive to flagellin (Table 2).

Another mAb was found concurrently with mAb F5C3. It was a phasin-like protein named mAb 3E6 that is reactive to a 21 kD B. pseudomallei protein, whereas mAb F5C3 is reactive to a 43 kD (Fig. 14A). The two-dimensional (2D) western blot analysis of B. pseudomallei lysate with mAbs F5C3 and 3E6 showed multiple reactive protein spots at roughly 43 kD and 21 kD respectively (Fig. 14B). A duplicate two- 27 dimensional (2D) SDS PAGE gel stained with SYPRO Ruby showed the total protein of

B. pseudomallei cells; the associated spots are shown by arrows (Fig. 14C).

A. B. C.

Figure 14. Western blot analysis and 2D western blot analysis identifying B. pseudomallei mAbs as flagellin and phasin-like protein. (A). Western blot probing B. pseudomallei lysate (strain 1026b) with hybridoma supernatant F5C3 and 3E6; reactive proteins are visible at 43 kD and 21 KD. (B). 2D Western blot probing B. pseudomallei lysate with mAbs F5C3 and 3E6 showing multiple reactive protein spots. (C). Duplicate 2D SDS PAGE gel was stained with sypro rubi; reactive proteins were excised from the gel for mass spectrometry analysis (Table 2).

Table 2. Mass spectrometry analysis identifying the corresponding spots

Sample Protein Name Accession No. Protein Rank Name Score C.I. % B03 1 flagellin [Burkholderia pseudomallei] gi|4581623 88.732 B04 1 flagellin [Burkholderia pseudomallei] gi|4581621 100 B05 1 hypothetical protein gsl1139 [Gloeobacter violaceus PCC 7421] gi|37520708 0 B06 1 flagellin [Burkholderia pseudomallei] gi|4581623 100 B07 1 putative heat shock protein [Bordetella parapertussis 12822] gi|33596095 0 B08 1 ABC transporter solute-binding protein [Bifidobacterium longum gi|23465745 0 NCC2705] B09 1 ketol-acid reductoisomerase [Burkholderia pseudomallei K96243] gi|53721340 100 B10 1 Precorrin-6x reductase CbiJ/CobK [Paracoccus denitrificans PD1222] gi|69937406 0 B11 1 putative dienelactone hydrolase [Burkholderia pseudomallei K96243] gi|53720896 91.452 B12 1 autonomous glycyl radical cofactor GrcA [Shigella flexneri 2a str. 2457T] gi|30063980 0

C01 1 phasin-like protein [Burkholderia pseudomallei K96243] gi|53719908 99.994 C02 1 phasin-like protein [Burkholderia pseudomallei K96243] gi|53719908 100 C03 1 phasin-like protein [Burkholderia pseudomallei K96243] gi|53719908 100 C04 1 phasin-like protein [Burkholderia pseudomallei K96243] gi|53719908 100

In addition, we further analyzed mAbs F5C3 and 3E6 reactivity by western blot.

The PVDF membrane was probed with mAbs F5C3 and 3E6 simultaneously. mAb F5C3

showed reactivity to flagellin at 43 kD from heat killed B. pseudomallei whole cell lysate

but failed to detect the protein in gamma irradiated B. pseudomallei. However, phasin-

like protein showed reactivity in both lanes at roughly 21 kD (Fig. 15; lane 1&2).

Moreover, we determined that mAb F5C3 bound to B. pseudomallei and B.

thailandensis but not to the closely related B. mallei. We probed both B. pseudomallei

and B. mallei with mAb F5C3; the result showed that B. mallei was not reactive with

mAb F5C3, whereas B. pseudomallei was reactive (Fig 15; lane 3&4). We demonstrated

mAb F5C3 bound to B. pseudomallei flagellin at an estimated 43 kD; however, B.

thailandensis flagellin was revealed at a lower molecular weight by the antibody (41 kD). 29

Nine species of B. cepacia complex bacteria were also probed with mAb F5C3, none of which were reactive (Data not shown).

Figure 15. The specificity of mAbs F5C3 and 3E6 determined by Western blot analysis. mAb F5C3 is reactive with flagellin (43 kD) from heat killed B. pseudomallei whole cell lysate (lane 1) but not reactive with gamma irradiated B. pseudomallei (lane 2). The membrane was probed with mAbs F5C3 and 3E6 simultaneously; phasin-like protein (21 kD) is reactive in both lanes. mAb F5C3 is not reactive with B. mallei whole cell lysate (lane 4), B. pseudomallei is shown in lane 3. mAb F5C3 appears to bind to B. pseudomallei flagellin at a slightly higher molecular weight than B. thailandensis flagellin (lanes 5 and 6, respectively). mAb 3E6 is reactive with B. mallei and B. thailandensis (lane 7 and 8 respectively).

Checkerboard ELISA was used to characterize the binding ability of mAb F5C3 to recombinant flagellin (rfliC) (Fig. 16). This checkerboard titration of rfliC against mAb

F5C3 in an ELISA format was performed to obtain the optimal dilution of both rfliC and mAb. Based on the results, the best conditions for binding mAb F5C3 to rfliC were at concentration1.3 µg/ml and mAb F5C3-labelled-HRP 0.8 µg/ml and alternatively at rfliC

0,63 µg/ml the mAb F5C3-labelled-HRP 2 µg/ml. 30

Figure 16. mAb F5C3 binds to rfliC as shown on the ELISA data. rFliC was serially diluted down a 96-well microtiter plate. Following a blocking and washing step, HRP- labeled mAb F5C3 was serially diluted across the plate.

Western blot analysis was used to determine the ability of mAb F5C3 to diagnose

B. pseudomallei infections. Five melioidosis patients’ urine samples were filtered to remove live bacteria. Therefore, whole cells were not present in the sample, only soluble bacterial antigens. Results indicated that mAb F5C3 could detect flagellin in 40% of urine samples of patients suffering from melioidosis and severe bacteremia. (Fig. 17).

Figure 17. Detection of flagellin with mAb F5C3 in urine samples from melioidosis patients. mAb F5C3 was reactive with flagellin in 2 from 5 urine samples (10 µl urine/well). As a positive control, B. pseudomallei lysate was probed (+). Urine from an uninfected control patient was used in lane 6. The table shows the amount of B. pseudomallei colony forming units (CFU) in the urine samples.

Table 3. summarizes the specificity of mAb 3C5, 4C7, and F5C3 to twelve species of Burkholderia genus and seven common pathogenic bacteria.

Table 3. The immunoreactivity of mAbs 3C5, 4C7, and F5C3

Bacterium Strain(s) mAb 3C5 CPS mAb 4C7 LPS mAb F5C3 Flagella immunoreactivity* immunoreactivity* immunoreactivity* B. mallei BEI resources ++ +++ - B. pseudomallei 1026b ++ ++ ++ B. thailandensis E264 - ++ +

B. multivorans HI2229 - - - B. vietnamiensis PC259 - - - B. dolosa AU0654 - - - B. ambifaria HI2468 - - - B. cenocepacia HI2718 - - - B. anthina AU1293 - - - B. stabilis HI2210 - - - B. pyrrocinia BC11 - - - B. cepacia BTS13 - - -

Pseudomonas ATCC 27853 - - - aeruginosa Streptococcus ATCC 10015 - - - pneumoniae Klebsiella pnuemoniae ATCC 13883 - - - Staphylococcus aureus ATCC 25923 - - - Enterobacter cloacae ATCC 23355 - - - Providencia stuartii ATCC 33672 - - - Escherichia coli ATCC 25922 - - - *-, No reactivity; +, low reactivity; ++, good reactivity; +++, highest reactivity.

Discussion

The genus Burkholderia is a group of closely related bacteria existing in soil and water; most of them are saprophytic organisms. However, at least eleven species of this group are important exceptions, such as B. pseudomallei and B. mallei, which are primary pathogens for humans and animals. In addition, nine species of the B. cepacia complex

(Bcc) are important opportunistic pathogens that cause serious infection in patients with cystic fibrosis (CF) (Weirsinga, et.al, 2006; Vandamme, et.al., 2007).

B. pseudomallei and B. mallei are both category B biothreat agents. B. pseudomallei is the causative agent of melioidosis, which is endemic in southeast Asia and northern Australia (Cheng and Currie, 2005; Dance, et.al., 1991). B. mallei is a facultative intracellular pathogen that causes glanders, an animal disease, that only accidentally infects humans. Those two bacteria are nearly genetically identical. The polysaccharide components present in these two species are quite homologous

(Vandamme, et.al., 2007).

The B. cepacia complex (Bcc) has been associated with a wide variety of infections, most often in patients with cystic fibrosis (CF) that are particularly susceptible to lung infections caused by these bacteria (Mahenthiralingam, et.al., 2005; Vandame, et.al., 2007). Bcc is divided into at least nine species because each species in this group has a similar phenotype but different genotypes (Mahenthiralingam, et.al., 2005). An interesting study shows that a CF patient who lives or travels to a melioidosis endemic area may also be vulnerable to pulmonary melioidosis; in addition, another report proved that patients with cystic fibrosis are at risk of pulmonary melioidosis. (Dance, 2002;

Holland et.al., 2002). 34

Another Burkholderia species that is closely related to B. pseudomallei is B. thailandensis. They are saprophytic bacteria found in water or soil and they coexist with

B. pseudomallei (Weirsinga, 2004). In contras to B. pseudomallei, B. thailandensis strains are not correlated with human disease and they are avirulent in the Syrian golden hamster animal model (Brett, et.al., 1997).

In this study, we examined a collection of monoclonal antibodies (mAbs), against

B. pseudomallei, and compared their reactivity on twelve Burkholderia species and eight other common pathogenic bacteria such as Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, and Eschericia coli. In order to produce a specific immunoassay that would identify melioidosis infection, we wanted to test if our mAbs were specific to B. pseudomallei so that they could be used as potential diagnostic tools for melioidosis.

We used bacteria from other genus of Burkholderia to characterize our mAbs because some studies have described a link between surface hydrophobicity of Gram- negative and Gram-positive bacteria and the potential to cause disease (Sarkar-Tyson, et.al., 2007). Moreover, many bacterial pathogens, such as Neisseria, Streptococcus,

Haemophilus, Klebsiella and Eschericia coli have been shown to be able to attenuate the bactericidal activity of complement and phagocytes by encapsulation with structurally diverse polysaccharides (Masoud, et.al., 1997)

Characterization of mAb 3C5

The mAb 3C5 was previously produced from Balb/c mice infected by whole- killed B. pseudomallei cells. By screening using western blot analysis, we determined that mAb 3C5 is specific to B. pseudomallei capsular polysaccharide (CPS) due to its 35 high molecular weight, proteinase-K insensitive and by the observation that it is does not bind to the CPS deleted mutant cells.

We characterized the mAb 3C5 with twelve species of Burkholderia genus and eight pathogenic bacteria by western blot analysis. The results showed that mAb 3C5 is only reactive to B. pseudomallei and B. mallei, but not to closely related B. thailandensis,

B. cepacia complex and common pathogenic bacteria. Similar to the Steinmetz et.al. study, we also found that monoclonal antibody against CPS were cross-reactive with B. mallei at a slightly lower molecular weight polysaccharide (Fig.3). This observation agrees with a previous report that demonstrated the B. mallei antigen has a lower molecular weight than the B. pseudomallei capsular polysaccharide (Anuntagool and

Sirisinha, 2002). A reasonable explanation for this observation is that B. mallei produces a similar polysaccharide. The genes of the CPS locus are > 90% identical between B. pseudomallei and B. mallei. CPS B. pseudomallei and B. mallei have the same chromosomal organization regions containing the capsule operon genes. However, B. thailandensis lacks 10 genes that have been truncated in that CPS region (Reckseidler-

Zenteno, et.al., 2009).

CPS has been considered as a potential candidate in serological diagnosis as well as a vaccine candidate. CPS is commonly found in the sera from a number of melioidosis patients. This observation suggests that the polysaccharide is immunogenic and could be a common constituent of B. pseudomallei (Masoud et.al., 1997).

Furthermore, we assumed that our mAb 3C5 is specific to CPS I, which is produced by B. pseudomallei. Reckseidler-Zenteno et.al. established the organization of the chromosomal regions containing the genes comprising the B. pseudomallei capsule 36 operon. He divided the Burkholderia CPS into four group; CPS I, CPS II, CPS III and

CPS IV; additionally, the CPS I is characterized to belong only to B. pseudomallei and B. mallei. However, B. thailandensis does not have CPS I, but has CPS II or exopolysaccharide (EPS) that is commonly present in the environmental organism, but not in the bacterial pathogen B. mallei (Reckseidler-Zenteno, et.al., 2009). That would suggest that mAb 3C5 is specific to distinguish melioidosis infection, since glanders that is caused by B. mallei and recognized with this mAb rarely occurs in human.

Western blot and fluorescence microscopy analysis of B. pseudomallei demonstrated that mAb 3C5 was reactive and bound to the B. pseudomallei capsule.

Furthermore, we tested mAb 3C5 with nine species in the B. cepacia complex and seven common pathogenic bacteria (Table 3). None of those bacteria were reactive with mAb

3C5; this indicates that mAb 3C5 could potentially be used as a specific diagnostic tool for melioidosis.

One member of B. cepacia group, B. stabilis, has been thought to have CPS because B. stabilis has a gene encoding the glycosyltransferases that was recognized in B. pseudomallei, B. mellei but not B. thailandensis using a subtractive hybridization assay

(Reckseidler et.al., 2001). However, although B. stabilis has a CPS encoding gene, our mAb 3C5 does not bind to B. stabilis. This finding supports the claim that mAb 3C5 is specific to the B. pseudomallei capsule.

Characterization of mAb 4C7

Our study showed that our mAb 4C7 was reactive to B. pseudomallei, B. thailandensis and B. mallei in a typical ladder pattern. Previous reports have demonstrated that B. pseudomallei, B. mallei and B. thailandensis are closely related 37 serologically (Anuntagool et.al., 1998). However, mAb 4C7 did not show any reactivity to nine species in the B. cepacia complex and to other common pathogenic bacteria that have been used in this study. We proposed that mAb 4C7 specifically targets the LPS in

B. pseudomallei.

A study revealed by Burtnick et.al. (2002) showed the DNA sequence of the B. mallei and B. pseudomallei O-polysaccharide (O-PS) gene cluster were identical, which contains 16 predicted ORFs. Additionally, the sequence alignment of the B. pseudomallei and B. mallei O-PS biosynthetic regions revealed 99% identity at the nucleotide level

(Burtnick et.al., 2002). This study supports our finding that mAb 4C7 is cross-reactive to

B. mallei. However, since glanders rarely cause disease in humans, we believe that mAb

4C7 could be used as a potential immuno-instrument to detect bacterial LPS in melioidosis patients.

In this study our mAb 4C7 reacted stronger to B. mallei than B. pseudomallei or

B. thailandensis. It seems that B. mallei has more epitopes to mAb 4C7 than B. pseudomallei and B. thailandensis, although this mAb was produced from B. pseudomallei. This indicates an epitope variation between the LPS of Burkholderia group. A study demonstrated that there were epitope variations among Burkholderia LPS.

That study showed that mAb specific to LPS B. cepacia strain had different binding patterns of LPS within and between the Burkholderia group (AuCoin, 2010). In addition,

The LPS B. mallei was previously shown to cross react with polyclonal antibodies raised against B. pseudomallei LPS; however, B. mallei LPS did not cross react with a monoclonal antibody specific for B. pseudomallei O-PS indicating that differences exist between B. mallei and B. pseudomallei O-PS (Burtnick et.al., 2002). In addition that 38 study also found that The O-antigen of B. mallei is slightly different; it does not produce acetyl modifications at the O-4 position of L-6dTalp.

The LPS of B. pseudomallei commonly exhibited a typical ladder pattern that is specific for B. pseudomallei LPS (Anuntagool, et.al., 2006). A previous study revealed that B. cepacia has a typical ladder pattern LPS (AuCoin et.al., 2010). Both

B.pseudomallei and B. cepacia have a typical ladder pattern; however, our mAb 4C7 was unreactive with B. cepacia. In addition, our mAb 4C7 did not bind to other species in B. cepacia complex or pathogenic bacteria as well. This indicated that although they have similar structure, the epitopes were not cross-reacted with each other.

Characterization of mAb F5C3

Based on the results, our mAb was designated as anti-flagellin B. pseudomallei by western blot analysis and mass spectrometry results. Firstly, we probe B. pseudomallei whole cells lysate with mAb F5C3, the mAb F5C3 bound to a 43 kD band in B. pseudomallei antigen. A previous study showed that the flagellin monomer proteins from four strains of B. pseudomallei (Pseudomonas pseudomallei) were determined to be approximately 43 kD molecular weight (Brett, et.al., 1994). In addition, the band disappeared when we treated the cells with proteinase-K, indicating that mAb F5C3 bound to a particular protein. To find the specific protein that the mAb was binding to, we performed a 2D SDS PAGE and Western blot analysis to aligned the associated protein spots. Through mass spectrometry analysis, we identified the protein that was reactive with mAb F5C3 as flagellin.

Some studies have debated the importance of flagella in causing disease. One study revealed that flagella were virulence determinant as their flagellin defective mutant 39 was avirulent for BALB/c mice when deposited intranasally. However, when challenged by intraperitoneal route, flagella were not required for virulence (Wikraiphat, et.al.,

2009). DeShazer et.al., also proved flagella or motility was not required in animal models of B. pseudomallei infection (DeShazer et.al., 1997).

We probe other Burkholderia species, including B. mallei, B. thailandensis and the B. cepacia complex with flagellin mAb, F5C3. Based on the results, we confirmed that B. mallei did not bind to flagellin mAb, but only B. thailandensis was reactive to mAb F5C3 by binding to a lower molecular weight band (roughly 41 kD). It has been known that B. mallei does not have flagella. However, B. thailandensis was reactive to mAb F5C3 with a lower molecular weight. The reasonable explanation for this observation would be that B. thailandensis has a 15 base pair (bp) deletion within the variable region of the flagellin gene fliC compared with B. pseudomallei (Chuaygud, et.al., 2008).

Conclusion

1. mAb 3C5 is reactive to a high molecular weight polysaccharide of B.

pseudomallei and B. mallei.

2. mAb 4C7 produces a ladder pattern (25-75 kD) and is reactive with B.

pseudomallei, B. thailandensis, and B. mallei.

3. mAb F5C3 is reactive to B. pseudomallei and slightly reactive to B. thailandensis

at roughly 43 kD.

4. mAb 3C5 and F5C3 may be used as a tool to develop a highly specific rapid

diagnostic test for melioidosis.

References

Adler, N.R.L., Govan, B., Cullinane, M., Harper, M., et. al. 2009. The molecular and cellular basis of pathogenesis in melioidosis: how does Burkholderia pseudomallei cause disease?. FEMS Microbiology Reviews: 33. 1079-1099.

Brent, A.J., Matthews, P.C., Dance, D.A., Pitt, T.L., and Handy, R. 2007. Misdiagnosing Melioidosis. Emerging Infectious Disease. February. Vol. 13(2). p. 349-351.

Brett, P.J., Mah, D.C.W. and Woods, D.E. 1994. Isolation and characterization of Pseudomonas pseudomallei flagellin proteins. Infection and Immunity. May. p. 1914-1919.

Brett, P.J., DeShazer, D., and Woods, D.E. 1997. Characterization of Burkholderia pseudomallei and B. pseudomallei-like strais. Epidemiol. Infect. 118, 137- 148.

Brett PJ, DeShazer D and Woods DE. 1998. Burkholderia thailandensis sp. nov., a Burkholderia pseudomallei-like species. Int J Syst Bacteriol;48 Pt 1:317-20

Burtnick MN, Brett PJ and Woods DE. 2002. Molecular and physical characterization of Burkholderia mallei O antigens. J Bacteriol;184:849-52

Charuchaimontri, C. et.al. 1999. Antilipopolysaccharide II: an antibody protective against fatal melioidosis. Clinical Infectious Disease: 29. p. 813-818.

Cheng, A.C. and Currie B. J. 2005. Melioidosis: Epidemiology, Pathophysiology, and Management. Clinical Microbiology Review. April. p. 383-416

Cheng, A.C., O’Brien, M., Freeman, K., et.al. 2006. Indirect Hemagglutination Assay in Patients with Melioidosis in Northern Australia. Am J Trop Med Hyg: 74. p. 330-334.

Chuaygud, T., Tungpradabkul, S., Sirisinha, S., et.al. 2008. A role of Burkholderia pseudomallei flagella as a virulent factor. Trans R Soc Trop Med Hyg; 102

Dance, D.A., Wuthiekanun, V., Naigowit, P., and White, N.J. 1989. Identification of Pseudomonas pseudomallei in Clinical Practice: use of Simple Screening Tests and API 20NE. Journal of Clinical Pathology.: 42. p. 645-648.

Dance, D.A.B. 1991. Melioidosis: the Tip of the Iceberg? Clinical Microbiology Reviews. Vol. 4. p. 52-60 42

Dance, D.A., Smith, M.D., Aucken, H.M., and Pitt, T.L. 1999. Imported Melioidosis in England and Wales. The lancet 353, 208.

Dance, D.A. 2002. Melioidosis. Curr. Opin. Infect. Dis. 15, 127-132.

DeShazer, D., Brett, P.J., Carlyon, R., and Wood, D.E. 1997. Mutagenesis of Burkholderia pseudomallei with Tn5-OT182: Isolation of Motility Mutants and Molecular Characterization of the Flagellin Structural Gene. Journal of Bacteriology; 179. April. p. 2116-2125.

Deshazer, D., Wagg, D., Fritz, D. & Woods, D. (2001). Identification of a Burkholderia mallei polysaccharide gene cluster by subtractive hybridisation and demonstration that the encoded capsule is an essential virulence determinant. Microb Pathog 30, 253–269.

Gan, Y.H., 2005. Interaction between Burkholderia pseudomallei and the host immune response: sleeping with the enemy. J. Infect. Dis. 192. 1845-1850

Gal, D., Mayo, M., Spencer, E., et.al. 2005. Application of a Polymerase Chain Reaction to detect Burkholderia pseudomallei in Clinical Specimens from Patients with Suspected Melioidosis. Am J Trop Med Hyg: 73. p. 1162- 1164.

Holden, M.T.G., Titball, R.W., Peacock S.J., et al. 2004. Genomic plasticity of the causative agent of melioidosis, B. pseudomallei. PNAS 101, 14240-14245.

Holland, D.J., Wesley, A., Drincovic, D., and Currie, B.J. 2002. Cystic fibrosis and Burkholderia pseudomallei infection: an emerging problem? Clin. Infect. Dis. 35, e138-140.

Inglis, T.J. et. al. 2000. Interaction between Burkholderia pseudomallei and Acanthamoeba species results in coiling phagocytosis, endamebic bacterial survival, and escape. Infect. Immun. Vol. 68. p. 1681-1686.

Jones, S.M., Ellis, J.F., Russel, P., et.al. 2002. Passive protection against Burkholderia pseudomallei infection in mice by monoclonal antibodies against capsular polysaccharide, lipopolysaccharide or proteins. J. Med. Microbiol. Vol 51. p. 1055-1062.

Kawahara, K., Dejsirilert, S., and Ezaki, T. 1998. Characterization of three capsular polysaccharides produced by Burholderia pseudomallei. FEMS Microbiology Letters, 169. p. 283-287.

Mahenthiralingam, E., Urban, T.A., and Goldberg, J.B. 2005. The multifarious, multireplicon Burkholderia cepacia complex. Nature Reviews Microbiology 3, 144-156.

Masoud, H., Ho, M., Schollaardt, T., and Perry, M.B. 1997. Characterization of the capsular polysaccharide of Burkholderia (Pseudomonas) pseudomallei 304b. Journal of Bacteriology. September. 5663-5669.

Nelson, J.W., Prior, J.L., Lever, M.S., et al. 2004. Evaluation of lipopolysaccharede and capsular polysaccharide as subunite vaccine against experimental melioidosis. Journal of Medical Microbiology: 53. p.1177-1182.

Nguay, V., Lemeshev, Y., Sadowwski, L., and Crawford, G. 2005. Cutaneous melioidosis in a man who was taken as prisoner of war by the Japanese during World War II. J. Clin. Microbiol.43, 970-972

Parry, C.M., Wuthiekanum, V., Hoa, N.T., et al. 1999. Melioidosis in southern Vietnam: clinical surveillance and environmental sampling. Clinical Infectious Disease, 29, 1323 – 1326

Peacock, S.J., Chieng, G., Cheng, A.C., et.al. 2005. Comparison of Ashdown’s medium, Burkholderia cepacia medium, and Burkholderia pseudomallei selective agar for clinical isolation of Burkholderia pseudomallei. Journal of Clinical Microbiology. Oct. p. 5359-5361.

Peacock, S.J. 2006. Melioidosis. Current Opinion Infectious Diseases. Vol 19. p. 421- 428.

Perry M.B., MacLean L.L., Schollaardt T., et al. 1995. Structural characterization of the lipopolysaccharide O antigens of Burkholderia pseudomallei. Infect. Immun 63:3348-3352

Pruksachartvuthi, S., Aswapokee, N., and Thankerngpol, K. 1990. Survival of Pseudomonaspseudomal; in human phagocytes. J. Med. Microbiol. Vol. 31. p. 109-114.

Puthucheary, S.D., Vadivelu, J., Ce-Cile, C., et. al., 1996. Short report: Electron Microscopic Demonstration of Extracellular Structur of B. pseudomallei. Am J Trop Med Hyg. 54: 313-314.

Reckseidler-Zenteno SL, DeVinney R and Woods DE. 2005. The capsular polysaccharide of Burkholderia pseudomallei contributes to survival in serum by reducing complement factor C3b deposition. Infect Immun;73:1106-15 44

Reckseidler-Zenteno, S., Moore, and Wood, D.E. 2009. Genetic and function of the capsules of Burkholderia pseudomallei and their potential as therapeutic targets. Mini-review in Medicinal Chemistry. 9, 265-271 Sarkar-Tyson, M., Thwaite, J.E., Harding, S.V., et.al. 2007. Polysaccharides and virulence of Burkholderia pseudomallei. Journal of Medical Microbiology 56. p.1005-1010.

Schaechter, M., Engleberg, N.C., Eisenstein, B., and Medoff, G. 1999. Mechanism of Microbial Disease. Third Edition. Lippincott Williams&Wilkins. p. 22-23.

Sirisinha, S., Anuntagool, N., Dharakul, T., et.al. 2000. Recent developments in laboratory diagnosis of melioidosis. Acta Tropica; 74. p. 235-245.

Steinmetz, I., Reganzerowski, A., Brenneke, B., et.al. 1999. Rapid identification of Burkholderia pseudomallei by latex agglutinatiom based on exopolysaccharide-specific monoclonal antibody. Journal of clin. Micro. January. 225-228.

Thepthai, C., Smithtikarn, S., Suksuwan, M., et.al. 2005. Serodiagnosis of melioidosis by a competitive enzyme-linked immunosorbent assay using a lipopolysaccharide-specific monoclonal antibody. Asian Pasific Journal of Allergy and Immunology; 23. p. 127-132.

Thomich, M., Herfst, C.A., Golden, J.W. and Mohr, C.D. 2002. Role of flagella in host cell invasion by Burkholderia cepacia. Infectious and Immunity. Vol 70(4). p. 1799-1806.

Vandamme, P., Govan, J., and LiPuma, J. 2007. Diversity and role of Burkholderia spp. Burkholderia molecular Microbiology and Genomics. Horizon Bioscience. p. 1-20

White NJ. 2003. Melioidosis. Lancet; 361:1715-22

Weirsinga, W.J., Poll, T.V., White, N.J., et al., 2006, Melioidosis: insights into the pathogenicity of Burkholderia pseudomallei. Nature vol. 4 April, p. 272 – 282.

Wuthiekanun, V., Anuntagool, N., White, N., and Sirisinha, S. 2002. Short report: A rapid method for the differentiation of Burkholderia pseudomallei and Burkholderia thailandensis. Am. J. Trop. Med. Hyg. 66(6). p. 759-751.

Wuthiekanun, V., Limmathurotsakul, D., Wongsuvan, G., et.al. 2007. Short report: Quantitation of B. pseudomallei in clinical samples. Am. J. Trop. Med. Hyg. 77(5). p. 812-813. 45

Yabuuchi, E., Y. Kosako, H. Oyaizu, I. Yano, H. Hotta, Y. Hashimoto, T. Ezaki, and M. Arakawa. 1992. Proposal of Burkholderia gen. nov. and transfer of seven species of the genus Pseudomonas homology group II to the new genus, with the type species Burkholderia cepacia (Palleroni and Holmes 1981) comb. nov. Microbiol. Immunol. 36:1251–1275.

Yan, K.T., Ellis, W.A., Mackie, D.P., et.al. 1999. Development of an ELISA to detect antibodies to a protective lipopolysaccharide fraction of Leptospira borgpetersenii serovar hardjo in cattle. Veterinary Microbiology: 69. p. 173-187.