# Proteins ofBartonella bacilliformis: Candidates for Vaccine Development

Authors: Cesar Henriquez-Camacho; Palmira Ventosilla; Michael F. Minnick; Joaquim Ruiz; Ciro Maguiña
Journal: International Journal of Peptides
 (2015)

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## Abstract

Bartonella bacilliformis is the etiologic agent of Carrión’s disease or Oroya fever. B. bacilliformis infection represents an interesting model of human host specificity. The notable differences in clinical presentations of Carrión’s disease suggest complex adaptations by the bacterium to the human host, with the overall objectives of persistence, maintenance of a reservoir state for vectorial transmission, and immune evasion. These events include a multitude of biochemical and genetic mechanisms involving both bacterial and host proteins. This review focuses on proteins involved in interactions between B. bacilliformis and the human host. Some of them (e.g., flagellin, Brps, IalB, FtsZ, Hbp/Pap31, and other outer membrane proteins) are potential protein antigen candidates for a synthetic vaccine.


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## Body

## 1. Introduction

Bartonella bacilliformis is a member of the alpha-2 subgroup of Proteobacteria and is the etiologic agent of Carrión’s disease or Oroya fever in humans. Bartonellosis has been historically described in Peru, Ecuador, and Colombia [1, 2], with Peru considered the most important endemic area in the world. This disease often, but not always, presents in a biphasic manner. The primary, life-threatening acute phase is characterized by a septic state worsened by a severe hemolytic anemia (bacteria invade the erythrocytes which are destroyed by the spleen) [3]. This phase, known as Oroya fever, has a high fatality rate (up to 88%) if not treated and is more common in children [4]. Fever, pallor, general malaise, myalgia, headache, jaundice, and hepato/splenomegaly are the main symptoms of Oroya fever [1]. Some patients develop, as a consequence of the septic state, a transient immunosuppression that results in the onset of opportunistic infections such as toxoplasmosis, tuberculosis, salmonellosis, shigellosis, histoplasmosis, malaria, and pneumocystosis [1, 5, 6]. In the secondary chronic phase, known as verruga peruana (Peruvian wart), the bacteria invade the endothelial cells producing wart-like hemangiomatous lesions of the skin and mucous membranes 4 to 8 weeks after the onset of Oroya fever. The duration of the eruptive phase is 3 to 6 months. Lesions are classified as miliary (small reddish papules < 3 mm in diameter), mular (nodular tumors > 5 mm), and diffuse subdermal nodules [7]. Inhabitants, especially schoolchildren, in endemic regions often develop the eruptive phase as the sole manifestation of the disease [8]. Humans are the only known natural reservoir forB.bacilliformis [7, 9].B.bacilliformis is believed to be transmitted to humans by bites of phlebotomine sand flies living in the high mountain valleys of the Andes [10].SinceB.bacilliformis was first described in 1905 by Alberto Barton; numerous research studies have been reported, including clinical and experimental studies. In the last 25 years, arguably the most important studies have been conducted to explain the physiopathology of the disease.The notable differences in clinical presentations during aB.bacilliformis infection suggest a complex adaptation to the human host with the objectives of persistence, maintenance of a reservoir state for vector-based transmission, and immune evasion. The bacterium-host relationship evokes a multitude of biochemical and genetic mechanisms involving both bacterial and host proteins.This review focuses on proteins involved inB.bacilliformis’ interactions with the human host. We hypothesize that certain proteins would be ideal protein antigen candidates to be included in a chemically synthesized vaccine to blockB.bacilliformis interactions with host cells (see Figure 1).Figure 1
Various protein antigens ofB.bacilliformis and their locations in the cell. Brp’s:Bartonella repeat proteins, FtsZ: cell division protein, Hbp’s: hemin-binding proteins, IalB: invasion-associated locus B protein, LppB: lipoprotein B, OM: outer membrane, PG: peptidoglycan, and CM: cytosolic membrane.Flagellin.B. bacilliformis cells are highly motile by lophotrichous flagella, appendages consisting of ~42 kDa flagellin protein subunits that are resistant to protease or trypsin treatment [11].B.bacilliformis was reported to possess 1 to 10 polar flagella 3 to 10 μm in length [11]. In addition to blood flow within the circulatory system, flagella provide the bacterium with a high degree of mobility and may serve to propel the pathogen during its search for erythrocytes [12].Bacterial motility seems to be required for internalization, since entry of nonmotileB. bacilliformis was not observed in the experiments of Mernaugh and Ihler [13].Antiserum to flagella significantly reducedB.bacilliformis association with red cells as compared to controls [11], and there was poor adherence of nonmotile variants and flagellin-minus (bald) mutants [14, 15]. These data suggest that flagella may possess adhesive qualities and/or they increase bacteria-host cell interactions [11]. In view of the significant decrease in erythrocyte infection due to antiflagellin antibodies, this protein is a promising antigen for use in a potential subunit vaccine.Brps.Bartonella species invade a variety of eukaryotic cells by employing trimeric autotransporter adhesion proteins (TAAs) [16]. The TAAs all use a type V secretion pathway and consist of a passenger domain and a beta domain used to deliver the passenger component out of the cell via the outer membrane.B.bacilliformis possesses threebrp genes encoding potentialBartonella repeat proteins (Brps) [17].B.bacilliformis Brp proteins share common domains and structural features with the TAAs. While the biological role ofB.bacilliformis Brp proteins has not yet been investigated, they may be involved in similar biological processes as the TAA proteins ofB.henselae andB.quintana, including autoagglutination, adhesion to host cells and extracellular matrix proteins (fibronectin and collagens), inhibition of phagocytosis, and induction of a proangiogenic response in host cells [10]. Immune-mediated inhibition of these hypothetical functions could possibly confer protection against the pathogen.IalB. The invasion-associated locus B (IalB) protein (see Figure 2) was originally discovered by screening aB.bacilliformis genomic library for erythrocyte-invasive strains of hostE.coli [18]. IalB has a significant effect on enhancingB.bacilliformis’ erythrocyte invasion. It is possible that environmental changes during passage of the bacterium from sand fly to humans (e.g., changes in temperature, iron availability, and pH) have an effect onialB gene expression [19]. In other research, IalB protein levels were correlated with changes inialB mRNA and changes of temperature/pH of the growth media. The greatest amount ofialB was present inB.bacilliformis that had been acid-shocked (pH 5), grown at 20°C, or temperature downshifted to 20°C from 30°C. As withialB mRNA levels, IalB protein amounts inB.bacilliformis decreased in response to basic pH (pH 8.0) or a temperature upshift to 37°C [20].Figure 2
Crystal structure of the invasion-associated locus B protein; 144 residues (77% ofBartonella bacilliformis IalB sequence) modeled with 100.0% confidence by the single highest scoring template. Program Phyre2 at http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index. Model based onB.henselae IalB crystal template c3dtdi_.Presumably,B.bacilliformis undergoes a temperature shift from 37°C to 20°C when it is ingested by a sand fly, as the blood meal in the insect cools to ambient temperature. Concurrent with this temperature downshift, bacterialialB expression would presumably be upregulated for erythrocyte invasion following transfer to another human host when the insect subsequently feeds. A change of the pH also occurs in the sand fly midgut (pH increases slightly to pH 7.4), whereialB expression would presumably be upregulated. When bacteria pass from the sand fly vector to a human host, the event would be signalled by a rapid upshift in temperature to 37°C and an increase in pH to 7.4. TheialB gene expression would be downregulated but not abrogated under these conditions. Undoubtedly, other bacterial factors are involved in erythrocyte adherence and invasion. IalB was localized to the inner membrane ofB.bacilliformis, where it is unlikely to directly interact with erythrocytes. In contrast, the IalB homolog ofB.henselae was localized to both inner and outer membrane fractions of the bacterium [21]. It is unknown if this protein is a transporter or signal transducer involved in human erythrocyte invasion, and its mechanism of action remains elusive [20].FtsZ. The FtsZ protein ofB.bacilliformis (FtsZBb) is a ~75 kDa protein and a structural homolog ofB.henselae’s cell division protein, FtsZ [20]. FtsZBb is strongly immunogenic in humans and was one of two major antigens (i.e., 75 kDa and 65 kDa proteins) identified in early work [9, 22]. In addition, the C terminus of FtsZBb contains a 256-amino acid region that is unusual and found in other related bacteria such asRhizobium meliloti. As a result, the FtsZBb is almost twice as large as the majority of the FtsZ homologs, perhaps contributing to its antigenicity [17]. Interestingly, theftsZ gene has been included in MLST schemes to study clonal and evolutionary relatedness ofB.bacilliformis strains [23].


## 2. Miscellaneous Outer Membrane Proteins

SeveralB.bacilliformis outer membrane proteins have been identified by a variety of means. Minnick identified 14 proteins ranging from 11.2 to 75.3 kDa. Three of these proteins (31.5, 42, and 45 kDa) were prominent immunoprecipitants when they were exposed to rabbit hyperimmune serum [24]. Iwaki-Egawa and Ihler identified sixB.bacilliformis proteins that mediate bacterial interactions with erythrocytes (100, 92, 84, 46, 37, and 12 kDa) [25].A 43 kDa immunogenic lipoprotein ofB.bacilliformis was identified through screening of a genomic DNA lambda library. The protein is a homolog to the LppB proteins ofHaemophilus spp. and the NlpD protein ofE.coli and was thus designated LppB [24]. The LppB lipoprotein is likely to use the same biosynthetic pathway as other bacterial lipoproteins. The protein’s prominent immunogenicity was demonstrated using five convalescent sera from patients with Oroya fever or verruga peruana [26].Buckles and McGinnis Hill demonstrated thatB.bacilliformis was able to bind to several human erythrocyte proteins, including α and β subunits of spectrin, band 3 protein, glycophorin A, and glycophorin B [27]. Band 3 is a major transmembrane glycoprotein of the erythrocyte membrane, and it may be one of the possible erythrocyte receptors ofB.bacilliformis [27]. In addition, band 3 has been suggested to be involvedPlasmodium invasion of erythrocytes [28, 29].


## 3. Hemin-Binding Proteins (Hbp’s)

TheB.bacilliformis hemin-binding proteins (Hbp’s) are homologs of the bacteriophage-associated, Pap31, protein ofBartonella henselae [30] and the five hemin-binding proteins ofB.quintana [31, 32].B.bacilliformis possesses three, tandemhbp-encoding genes in its chromosome. In addition to serving as a receptor for hemin and associating with the phage coat, data from work withB.henselae suggest that these proteins may also serve as adhesins for fibronectin, heparin, and host cells such as endothelial cells [33]. The Hbp (Pap31) proteins are highly expressed antigens in growing cultures ofB.bacilliformis and are immunologically dominant, making them ideal antigens for use in enzyme-linked immunosorbent assays (ELISAs) and in immunoblots. In fact, use of this single protein antigen permits the use of ELISA, Western blot methods, or rapid lateral flow assay for the diagnosis of aB.bacilliformis infection [34]. To what extentB.bacilliformis Hbp’s are immunoreactive in humans is unknown. However, the HbpE homolog ofB.quintana is a commonly recognized antigen in sera from trench fever patients [35]. This observation suggests that one or moreB.bacilliformis Hbp’s may also be seroreactive in humans and thus useful subunit vaccine candidates.


## 4. Conclusions

B.bacilliformis infection represents an interesting model of human host specificity by a bacterium. While we now know more about the biology of the bacterium and the pathogenesis of Carrión’s disease, we still lack adequate knowledge to select specific protein candidates to generate a subunit vaccine formulation. Studies regarding comparative genomics and proteomics are sorely lacking, especially considering the significant impact that bartonelosis outbreaks represent for the affected populations in South America. Carrión’s disease is an emerging infectious disease that is not amenable to eradication with antibiotics or pesticides. Thus, the most effective intervention strategy to improve public health will be the development of a vaccine. Advances inB.bacilliformis genomics and proteomics hold the promise a rational vaccine design in the near future [36].



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