Pasteurella Multocida: Composition, Genetics and Biosynthesis of Capsular Polysaccharides (CPS) of Serotypes A, B, D, E and F

Pasteurellaceae is a family, initially proposed by Pohl in 1979 (Foster et al., 2000), which represents the commensals or vertebral parasites, unable to survive well outside the natural host. Through the application of advance genetic based taxonomy and identification methods, the family of Pasteurellaceae is now consisting of 28 genera since 1995, including the genus, Gallibacterium. Recently, Actinobacillus, Pasteurella and Haemophilus have also been added in this family as new genera (Eid et al., 2019; Fenwick and Rycroft, 2022; Chong et al., 2021). There are so many devastating and common pathogens that include Actinobacillus pleuropneumoniae, Pasteurella multocida, Avibacterium paragallinarum and Mannheimia haemolytica. Pasteurella genus contains coccobacillary to rod shaped bacteria whose width ranges from 0.3–1.0 μm while their length ranges from 1.0–2.0 μm (Peng et al., 2019; Boyce et al., 2004). They can be grown under aerobic, anaerobic and even under the microaerophilic condition at temperature ranging from 37 ℃ to 41 ℃. This genus is currently containing 12 species (Boyce et al., 2010).

It is a coccobacillus, non-motile and penicillin sensitive gram-negative bacterium (Zhao et al., 2021; Smith et al., 2021). It was first identified in a bird infected by cholera in 1878 and successfully isolated in 1880 by a French microbiologist named Louis Pasteur. This bacterium was named after the name of the scientist (Peng et al., 2019). There are four subspecies of P. multocida which include the type strain: Multocida, septica, gallicida and tigris (Harper et al., 2006). P. multocida strains are classified into serogroups or serotypes (A, B, D, E and F) on the basis of capsular antigens. This system was developed by Carter in 1955 and launched for the classification in 1963 based on haemagglutination (Christensen et al., 2022; Carter, 1955). They are also categorized into 1–16 serotypes which are based primarily on lipopolysaccharide (LPS) antigens (Smith et al., 2021; Michael et al., 2021).

P. multocida is a major pathogen among the members of Pasteurella, responsible for many animal diseases such as bovine hemorrhagic septicemia, swine atrophic rhinitis, and avian fowl cholera and severe economic losses in poultry and cattle industry (Zhao et al., 2021; Wilkie et al., 2012; Xiao et al., 2021). The processes by which these bacteria infect the mucosa, elude the innate immunity, and cause the systemic illnesses are being explained slowly. Capsule and lipopolysaccharide have been identified as the key virulence factors so far (Jiménez-Guerra, 2024). The capsule appears to have a role in bacterial phagocytosis evasion and complement resistance, whereas full lipopolysaccharide is required for bacterial survival in the host (Khamesipour et al., 2014). P. multocida toxin (PMT), putative surface adhesions, and iron acquisition proteins are among the additional virulence components discovered by both directed and random mutagenesis (Hashimi et al., 2023). Many critical virulence factors, including those necessary for adhesion and penetration of host cells, as well as for survival in a poorly nutritious and hostile environment, are likely to remain unknown (Harper et al., 2006). These diseases are associated with the particular serotype/es.

Hemorrhagic Septicemia (HS) majorly affects the livestock animals such as cattle, buffalo, camel and pigs (De Alwis, 1999; Mushtaq et al., 2022) predominantly infected either by serotype B:2 (Asian and European serotype) or serotype E:2 (African serotype) of P. multocida (Almoheer et al., 2022; Boyce et al., 2000). The observed clinical signs in affected animals are the elevation of temperature ranging from 102 to 107oF, chest and neck swelling, salivation, inappetence and protrusion of tongue. Sometimes, development of tympany, eye discharges and oedema have also been observed (Sheikh et al., 1996; Hassan and Mustafa, 1985; Asghar et al., 2023).

Progressive Atrophic Rhinitis (AR) is particularly caused by specific toxins produced by P. multocida serotype A and/or D i.e. PMT (Peng et al., 2019; Boyce et al., 2000; Guan et al., 2023). Major clinical symptoms include: nasal crusting, mucosal atrophy, paradoxical feeling of nasal congestion, decreased growth, facial distortion, enlarged nasal space and fetor while epistaxis, sinusitis and anosmia are also accompanied with these symptoms (Liva et al., 2021).

Fowl Cholera or FC, also known as avian cholera, is a serious septicemic contagious disease of poultry and wild birds that can either be present in acute or chronic form (Omaleki et al., 2022) and its etiological agents are P. multocida strains (especially serovar A:1, A:3, A:4 and serovar D to a lesser extent) (Peng et al., 2019; Saha et al., 2021). Generally, acute FC is caused by the serotype A whose symptoms include anorexia, fever, ruffled feathers, diarrhoea, oral mucous discharge and increase in breathing rate (Harper et al., 2006; Reuben et al., 2021). Apparent condition of the animals suffering from HS, AR and FC has been shown in the Figure 1.

In Figure 2, there is a visualization map of authors from 2010 to 2025 and this map was made by VOS viewer (version 1.6.20). Data was extracted using the Dimensions database using the terms, “Pasteurella multocida AND Capsular polysaccharides AND composition AND genetics AND biosynthesis”. The map shows the clusters of authors who frequently contribute and collaborate in the composition, genetics, and biosynthesis of CPS of P. multocida. Authors in yellow indicate recent active involvement in the research field.

In Figure 3, there is a visualization map of countries that have contributed so far. This map is made by VOSviewer. Data was extracted using the Dimensions database using the terms, “Pasteurella multocida AND Capsular polysaccharides AND composition AND genetics AND biosynthesis”. The map shows the clusters of countries who frequently collaborate in the composition, genetics, and biosynthesis of CPS of P. multocida. Countries in yellow indicate recent active involvement in this area of research. United States stands out as a central node with numerous connections, indicating its significant contribution. Moreover, Canada, Australia, Germany, India, and China are also leading in this research field.

Virulence is the measure of the pathogenicity of an organism. Virulence factors or pathogenicity factors are the molecules, cellular structures or regulatory systems which enable the microorganisms for colonization, immune-evasion, immunosuppression, getting nutrients and entry/exit causing the virulence in host cell ultimately (Argüello-García and Ortega-Pierres, 2021). There are various virulence factors in P. multocida, involved in its pathogenicity (Casadevall and Pirofski, 2009) including CPS, LPS, toxins, fimbriae, adhesins, iron regulating and iron acquisition proteins, hyaluronidase and several outer membrane proteins (Boyce et al., 2010; Kubatzky, 2012; Dabo et al., 2007; Van Nguyen et al., 2023; Cao et al., 2024). Studies have shown that capsule of P. multocida is very important in the development of vaccines. A lot of research is being carried out to develop the vaccines to prevent the deaths of livestock especially in the developing countries. Due to the importance of capsular polysaccharides (CPS) and their diversity in different serotypes, they are being studied well (Somarajan et al., 2007).

Bacterial capsule is a large polysaccharide structure lying outside the cell and can be a cause of many diseases (Peterson, 1996; Hathaway et al., 2016). It is not confined to gram negative bacteria but also present in gram positive bacteria. In contrast to slime, capsule is thicker, tightly bound to the cell wall, well organized, difficult to be washed off and consists only of polysaccharides (Baselga et al., 1994). Capsule and slime are sometimes collectively called as glycocalyx (Weinbaum et al., 2007). Monosaccharides are joined together through glycosidic linkages to make capsular polysaccharide (Weinbaum et al., 2007). Bacteria containing capsule are known as polysaccharide encapsulated bacteria (Yuan et al., 2021) and some of these bacteria make smooth or rough colonies on agar media (Orynbayev et al., 2019). P. multocida also contains capsule whose chemical composition varies in its different serotypes (A, B, D, E and F) (Rimler and Rhoades, 1987).

Capsule is an important virulence factor which means that it helps bacteria to cause disease by protecting them from phagocytosis by macrophages (Daffe and Etienne, 1999). As it contains water so it also protects from desiccation (Tipton et al., 2018; Reckseidler-Zenteno, 2012). Capsule is also important for the structural integrity and controlling the flow of nutrients. It may also act as a signal for the host to produce the needed nutrients. Capsule also helps in the adherence of bacterial cells with each other and to non-living surfaces as well.

CPS itself and its conjugate with other proteins have a strong potential for vaccine development. It has been observed that although CPS is essential for virulence, the antibody responses elicited against CPS are not necessarily the primary mediators of protective immunity (Kadioglu et al., 2008). Instead, other surface-exposed proteins, such as outer membrane proteins (OMPs) and lipoproteins, may serve as better vaccine targets (Hara and Nathan, 2022). Indeed, several recombinant protein vaccines targeting OMPs (e.g., PlpE and OmpH) have shown promising results in preclinical studies (Zhou et al., 2023; Jogi et al., 2023).

The challenge in developing an effective CPS-based vaccine for P. multocida lies in the intrinsic T cell-independent nature of polysaccharide antigens. Such antigens typically generate a short-lived immune response with poor immunological memory. To overcome this limitation, conjugate vaccines (chemically linked CPS to a protein carrier) have been developed for other pathogens, significantly improving immunogenicity. However, for P. multocida, the high variability in capsule composition across serogroups (A, B, D, E, and F) poses an additional hurdle for designing a broadly protective CPS-based vaccine (Hara and Nathan, 2022). This serogroup heterogeneity means that a vaccine targeting the capsule of one serogroup might not protect against another. Recent work has identified novel surface lipoproteins such as PmSLP, which are highly conserved among bovine isolates and can elicit robust and long-lasting immune responses in both mice and cattle (Fegan et al., 2024).

Although, most of the P. multocida strains produce the capsule made up of polysaccharides, different strains with different chemical composition of capsule have been identified (Rimler and Rhoades, 1987).

Serotype A has capsule primarily made up of hyaluronic acid (HA) or hyaluronan which is a linear polysaccharide consisting of β-D-glucuronic acid (β-D-GlcA) and β-D-N-acetylglucosamine (β-D-GlcNAc) repeating units joined together with the help of alternating β-1,4 and β-1,3 glycosidic linkages (Pasomboon and Chumnanpuen, 2021; Rosner et al., 1992; Smallman et al., 2022). Its chemical structure has been shown in the Figure 4.

Previous studies: The exact chemical composition of capsular polysaccharides of serotype B and E had not been determined till 2021 (Pasomboon and Chumnanpuen, 2021). According to the previous studies it was shown that the capsule of serotype B consists of mannose (Man), arabinose (Ara) and galactose (Gla) with the ratio of 2.0:0.5:0.8. The molecular weight was found to be 90 kDa in ziwittergent buffer by gel filtration and 900 kDa in Phosphate Buffer Saline (PBS) showing that it exists in decamer form in its native state (Pasomboon and Chumnanpuen, 2021). On the other hand, chemical composition of capsule of serotype E remained totally unclear till 2021.

Latest studies: Recently, the capsular polysaccharides of serotypes B and E have been determined and compared with respect to their structures, genetics and serology. Full nuclear magnetic resonance (NMR) spectroscopy revealed that there is a high similarity between CPS repeat units of both serotypes with minor differences. In serotype B, mannosaminuronic acid (ManNAcA) is substituted by fructose (Fruc) at the position 3 and by GlcNAc at position 4. In serotype E, linkages are reversed. At position 3, ManNAcA is substituted by GlcNAc and by fructose at position 4. The presence of glycine in the capsular polysaccharide of serotype B is a unique characteristic (Michael et al., 2021; Richardson et al., 2023). Chemical composition of capsule of serotype B and E has been shown in the Figures 5, 6.

Serotype D has capsule made up of heparosan (Guan et al., 2019) which is a linear polysaccharide consisting of β-D-glucuronic acid (β-D-GlcA) and α-D-N-acetylglucosamine (α-D-GlcNAc) repeating units joined together with the help of 1,4 glycosidic linkages (DeAngelis et al., 2002) as shown in the Figure 7.

Serotype F has capsule made up of chondroitin (Guan et al., 2019) which is a linear polysaccharide consisting of β-D-glucuronic acid (β-D-GlcA) and β-D-N-acetylgalactosamine (β-D-GalNAc) repeating units joined together with the help of 1,3 glycosidic linkages with alternating units linked by 1,4 glycosidic linkages (DeAngelis et al., 2002) as shown in the Figure 8.

Genetic sequences of all genes of all five serotypes have been determined that are involved in the capsular biosynthesis (Chung et al., 1998) and by comparing their genetic organization with other gram-negative bacteria it has been shown that there is similarity of these sequences with group II capsule biosynthetic operons (Boyce et al., 2000). The genes can be grouped into three regions (R1-3) (Guan et al., 2019).

The first group (Region 1) produces a set of proteins that function as a delivery system, transporting essential materials to the cell’s surface. Regions 1 is highly conserved among all the serotypes of P. multocida and encode proteins to make ATP binding cassette transport system (a protein complex) (Boyce et al., 2010). This transport system plays role in the export of polysaccharides to the surface of bacteria. Role of hex in serotype A:1 and role of cexA in serotype B:2 in the transport of polysaccharides to the surface have been determined and confirmed by mutagenesis (Harper et al., 2012).

The second group (Region 2) contains genes that act like construction workers, piecing together the sugar building blocks to form the capsule. Region 2 consists of the genes that encode for the synthases for the polymerization of serotype specific polysaccharides. Synthase PmHAS is responsible for the polymerization of hyaluronic acid, synthase PmHS1 is responsible for the polymerization of heparin and synthase PmCS is responsible for the polymerization of chondroitin in serotypes A, D and F respectively (DeAngelis et al., 1998; Kane et al., 2006). Roles of these synthases have been confirmed by mutagenesis and/or functional analysis. These synthases as bifunctional transferases can add both glucuronic acid and N-acetyl glucosamine or N-acetyl galactosamine in growing polysaccharide molecule. Mutagenesis has also confirmed the role of bcbH encoded protein in type B polysaccharide synthesis (Boyce and Adler, 2001).

The third group (Region 3) makes sure the capsule is properly attached to the bacterial cell, much like an anchoring system. This coordinated process is crucial for the bacterium’s ability to protect itself and cause disease. Region 3 is also highly conserved though all the serotypes of P. multocida consisting of the genes which encode proteins responsible for lipidation as well as surface attachment of polysaccharides (Boyce et al., 2010). R3 has two genes; phyAB and lipAB. These genes encode proteins that are responsible for the substitution of phospholipids for the anchorage of CPS (Roberts, 1996). However, in serotypes B and E, loci lipA is located adjacent to Region 1 for the diagrammatical representation of the synthesis of capsular polysaccharide (Harper et al., 2012). Genetic organization of capsule biosynthetic loci of all serotypes of P. multocida has been shown in the Figure 9.

Later on, outside the capsule biosynthetic region, a cryptic heparin synthase gene was discovered in serotype D which encodes heparin synthase. Homologous genes were also discovered in other A, D and F serotypes. It was proposed without any experimental investigation that these synthases may give variations in capsule composition (DeAngelis and White, 2004).

The development of the typing of P. multocida with the help of multiplex PCR based on the gene sequences present in region 2 was facilitated by the clarification on the genetic basis for capsule biosynthesis (Townsend et al., 2001). As this PCR-based typing is much easier and reliable so this has replaced the conventional serotyping and now is being used as a gold standard throughout the world (Arumugam et al., 2011). Functions of ORFs of different serotypes has been summarized in the Table 1.

Table 1: Function of ORFs of P. multocida.

Table 2: Summary of CPS composition, genetics and virulence in serotype A, B, D, E, and F.

Genetic organization is divided into three distinct regions: Region 1, which is highly conserved and encodes components of the ATP-binding cassette (ABC) transport system responsible for polysaccharide export; Region 2, which contains the genes encoding serotype-specific synthases that catalyze the polymerization of sugar units into the growing capsule; and Region 3, which comprises genes involved in the lipidation and surface attachment of the capsule.

Summary of CPS composition, Genetics and Virulence in Serotype A, B, D, E, and F has been shown in Table 2 for a holistic understanding.

The capsular glycosaminoglycan (GAG) of P. multocida is produced in the inner membrane of bacteria and then transported to outside through the ATP binding cassette (ABC) transporter which belongs to the translocases and is present from prokaryotes to humans (Willis and Whitfield, 2013a). Synthesized glycolipids attach to the cell wall by making hydrogen bonds. The biosynthesis of CPS involves initiation of GAG synthesis, extension of GAG disaccharide units and export of GAG (Willis and Whitfield, 2013b).

In addition to the CPS based vaccine development, we also need to extensively study the CPS biosynthesis and the enzymes involved in this biosynthesis to find potential drug targets. Small molecules blocking PmHAS (serotype A), PmHS (serotype D), or PmCS (serotype F) could prevent capsule assembly. Compounds targeting HexA/CexA (ATP-binding proteins in R1) could halt CPS export, rendering bacteria susceptible to host defenses.

In the initiation, GAG glycolipid terminal is synthesized where three steps are involved. (I) PhyB (glycosyltransferase) similar to E. coli protein KpsS, transports the first eight carbon sugar, β-3-deoxy-D-manno-oct-2-ulosonic acid (β-KDO) to lysophosphatidylglycerol (lyso-PG) receptor (Chung et al., 1998; Willis and Whitfield 2013a, b; Ovchinnikova et al., 2016). (II) PhyA (glycosyltransferase) similar to E. coli protein KpsC, adds five to nine β-KDOs to synthesize poly-β-KDO linker chain (Willis and Whitfield 2013a, b). (III) HyaE transports and adds UDP sugar molecules to poly-β-KDO linker chain and this is the first residue of glycolipid terminal (Willis and Whitfield 2013a). So, the bacterium starts by building a “starter anchor” made of sugars and fats (a glycolipid). This anchor acts like a foundation for the capsule. Structure of glycolipid has been shown in the Figure 10.

The extension or polymerization of GAG disaccharide units is essentially similar in serotypes A, D and F (DeAngelis and White, 2004; DeAngelis, 1996; DeAngelis and Padgett-McCue, 2000). PmHAS in serotype A, PmHS in serotype D and PmCS in serotype F act as bifuntional glycosyltransferase which catalyze the transfer of both β-D-acetylglucosamine (β-D-NGlcNAc) and β-D-N-acetylgalactosamine (β-D-GlcUA) using 2 transferase active sites. These proteins add the activated sugar molecules to the glycolipid terminal, release UDP and form the polysaccharide of the relevant serotype (DeAngelis 1999; Tracy et al., 2007).

Here, n is the degree of polymerization which is 20-100 and 103-104 in serotypes D and F, respectively.

In simple words, enzymes add sugar molecules to the anchor. These sugars are carried into place by UDP molecules acting as a “delivery truck”. The enzymes link them into long chains.

The export of GAG outside the cell surface mainly depends upon the ABC transport system (Willis and Whitfield, 2013a). The encoded protein products of first four genes (Region 1) together make an ATP transport system. HexA encoded by hexA, as an ATP binding protein delivers the energy for the GAG transportation. HexB encoded by hexB, is the protein of inner membrane. HexC encoded by hexC, is the protein of inner membrane that has a periplasmic domain. HexD encoded by hexD, is

the protein of outer membrane (Chung et al., 1998). GAG chain passes through the inner membrane, periplasmic space and outer membrane via a channel made by HexB/C/D with the help of ATP binding domain and ultimately makes the covalent bond with phospholipid at the cell surface (Roberts, 1996). Mechanism of biosynthesis of capsule has been shown in the Figure 11.

The process is depicted in three main stages. Initiation: A glycolipid precursor is synthesized by the sequential addition of β-KDO units to a lipid receptor, laying the foundation for capsule assembly. Extension: Bifunctional glycosyltransferases (such as PmHAS, PmHS, or PmCS, depending on the serotype) add sugar units, forming a polysaccharide chain by polymerizing glycosaminoglycan (GAG) disaccharide units. Export: The fully assembled capsule is translocated to the bacterial surface through the action of the ABC transporter system, which ensures its proper attachment.

Regulatory sequences: Expression of capsular biosynthetic loci depends upon the operon. Contrary to R3, R1 and R2 genes make a single transcriptional unit because they undergo the transcription in the same direction. The promoter of both R1 and R2 exists in between R2 and R3 (Boyce et al., 2000, 2010). In serotype A, R2 and R3 promoter is present between phyA and hyaE that is recognized by a transcription factor, σ70. -35 box and -10 box in the promoter are separated from each other by 17bp. R1 and R2 transcription initiation site is present at the 37bp upstream of start codon of hyaE (Steen et al., 2010), while ribosome binding site (RBS) is at 8bp upstream to stop codon of hexA and 4bp downstream of stop codon of hexA. Regulatory sequences of R3 are not well known yet however this is supposed to be not involve σ70 (Guan et al., 2019; Chung et al., 1998).

Transcriptional regulation: There is a factor for inversion stimulation (Fis) protein, which performs the transcriptional regulation and regulates the expression of capsule (Steen et al., 2010; Dorman and Deighan, 2003). This nucleoid associated protein is composed of 99 amino acids and has two of its biding sites in promotor region. From 73 to 94 amino acids, there is a DNA binding motif that can bind with promoter region and positively regulate the GAG genes (Steen et al., 2010; Bagchi, 2015). If mutation occurs in Fis gene, it causes production of acapsular strains even in the presence of intact nucleotide sequences. Fis gene is not always present in the capsular gene cluster in all serotypes. This gene also regulates the transcription of some other virulence factors for example LPS, Pasteurella Lipoprotein E (plpE) etc. (Steen et al., 2010).

There is a protein named as host factor for Qβ (Hfq) which is an RNA chaperone protein (Møller et al., 2002) encoded by hfq gene and regulates the translation of capsule biosynthesis loci (Mégroz et al., 2016; Carmichael, 1975). This protein together with GcvB (a small non-coding RNA molecule) as an Hfq-GcvB complex, targets the mRNA transcribed by capsular biosynthesis gene and positively regulates the gene expression (Gulliver et al., 2018). Hfq-GcvB complex, binds with the target mRNA via specific seed sequence, opens the secondary structure and exposes the RBS and AUG (start codon) (Gulliver et al., 2018; Fröhlich and Vogel, 2009). Ribosome then binds with RBS and AUG, activates the inhibited mRNA and initiates the translation. The seed sequence which is present on mRNA is 5’-AUGUUGUGUU-3′ while the complementary sequence present on GcvB is 5’-AACACAACAU-3′ (Gulliver et al., 2018). Mechanism of regulation of biosynthetic loci of P. multocida has been shown in the Figure 12.

The figure highlights both transcriptional and translational control mechanisms that modulate the expression of capsule biosynthetic genes. The binding of general transcription factors such as σ70 and regulatory proteins like Fis to specific promoter regions initiates and modulates the transcription of the gene clusters. The interaction of the RNA chaperone Hfq with the small RNA GcvB facilitates the exposure of ribosome binding sites on the mRNA, enhancing translation.

P. multocida has many capsular serotypes i.e. A, B, D, E, F whose capsules are made up of hyaluronic acid, mannosaminuronic acid, heparosan, mannosaminuronic acid, and chondroitin respectively. R1 of capsular biosynthetic gene locus encodes proteins to make ATP binding cassette transport system while R2 and R3 are responsible for the polymerization of polysaccharides and lipidation of polysaccharides respectively. CPS is synthesized by the initiation, extension and export of GAG units which is regulated by many genes at transcriptional and translational level.

This review article has gathered all the recent informations and studies about the composition, genetics, and biosynthesis of CPS of all serotypes of P. multocida which we do not find in any other article so that we can find the solutions of related diseases in animals.

Waqar Siddique wrote this article under the guidance of Sadia Mahboob and Waqar Rauf. Sobia Jabeen and Zubera Naseem helped in drawing the chemical structures of CPS using ChemDraw. Fiza Shafaqat helped in reviewing the article and mentioned the areas of improvement.

The authors have declared no conflict of interest.