A focus on virulence factors, regulation, comparative genomics, and

clinical implications for B. Pertussis

Abstract: Bordetella Pertussis is a bacterium which is responsible for the disease pertussis or whooping

cough. This paper reviews laboratory and clinical evidence regarding its mechanism of action, its

virulence genes in various strains, and the efficacy of some major Pertussis vaccines. Clinical studies

indicate that as B. Pertussis continues to mutate, vaccines are becoming less effective in treating the

population. Insight into the possible mutations and effects thereof are critical in providing a better

defense for the public health of the population.

Keywords: Bordetella Pertussis, Review, Clinical, Genomics, Virulence, Toxin, Comparative Genomics, Regulation

I. Virulence factors Virulence Factors Invasion Process

Fedele, G., et al.1 examines the virulence factors of B. Pertussis. About 40 million whooping cough cases and about 200,000 to 400,000 pertussis deaths are recorded each year. B. Pertussis continues to circulate even in populations with good vaccine coverage for children due to the degrading protection of vaccination.

Virulence factors of B. Pertussis which are involved in bacterial adhesion and

invasion by altering the local cell environment include: pertussis toxin, tracheal

cytotoxin, adenylate cyclase toxin, filamentous hemagglutinin, and the

lipooligosaccharide. Adhesins include filamentous hemagglutinin (FHA), pertactin

(PRN), and fimbriae (Fim).

After initial infection with B. Pertussis, humans produce antibodies to the

virulence factors, including PRN and Fim. Anti-Fim antibody after vaccination with

complete pertussis vaccine was found to correlate to the efficacy. A correlation was

found between vaccine protection and the presence of anti-PRN, anti-Fim, and anti-PT

antibodies in the serum. Many of the virulence factors have been included in vaccines

such as PT, FHA, PRN, and Fim. PT is the most complex virulence factor expressed by

B. Pertussis, composed of five different subunits, named S1 through S5 according to

decreasing molecular weights.

The A subunit is an ADP-ribosyltransferase and represents S1. The B subunit,

called the B-oligomer is a pentameric ring composed of S2, S3, S5, and two S4

subunits which is responsible for binding the toxin to the target cells. After binding of the

toxin to target cells, the B-oligomer helps in the transport to the Endoplasmic reticulum,

where the S1 travels into the cytosol. The A-protomer becomes activated and adds

ribosomes to specific target proteins to disrupt signaling pathways and induce multiple

downstream effects.

Affected cells include the β cells of pancreatic islets, adipocytes, macrophages,

lymphocytes, and other cells that cause sudden symptoms and neurological issues. The

toxin may also cause hyperinsulinemia, hypoglycemia, and changes in histamine

sensitivity.

PT may be the cause of sudden coughing but it is still widely debated. Rodent

studies indicated a role for PT in inducing coughing in pertussis. However, human

infections with B. parapertussis which does not express PT still have identical sudden

coughing. This may be due to an additional infection with B. Pertussis after onset of B.

parapertussis infection. PT exhibits its effects in more ways than through enzymatic

activity. The B-oligomer of PT binds to multiple signaling receptors expressed on the

cell surface, such as platelet glycoprotein and components of the T cell receptor. PT

plays a critical role in the local infection, mostly by modulation of the host immune

response to B. Pertussis.

Parton, R.2 generates host specificities and virulence factors of important B.

Pertussis strains adapted below:

Table 1: Host Specifics of various Bordetella species

Adapted (changed man to human) from Parton, R. (Need permission to use)

Table 2: Virulence factors of major Bordetella species

Taken from Parton, R. (Need permission to use)

Function of core virulence factors

Fedele, G., et al.3 overviews virulence factors of B. Pertussis which can alter the

cell environment and adhere including: pertussis toxin, tracheal cytotoxin, adenylate

cyclase toxin, filamentous hemagglutinin, and lipooligosaccharide.

Pertussis toxin (PT), produced by Bordetella Pertussis, is a toxin which binds to

most mammalian cells and targets specific G proteins, thus inhibiting the ability of the

target G protein to function in signaling pathways. The effects caused by PT can vary

depending on which G protein is inhibited. PT plays a role in the onset of infection by

suppressing host immune responses to Bordetella Pertussis.

Adenylate Cyclase Toxin (ACT) is another critical toxin involved in B. Pertussis.

When the lung infection has begun, ACT interacts with tracheal epithelial cells, inserting

itself through cytoplasmic membranes, which aids in the ability of the bacteria to adhere

to the lining of the airway. Host phagocytes attempt to respond to the infection site but

are disabled by the cAMP generated by ACT. This toxin is unique in its ability to cross

cell membranes and exist within the cytosol.

Liposaccharides (LPS) represent an interesting endotoxin which lacks the typical

O polysaccharide or O antigen chain forming the outer domain of LPS. A non-repeating

trisaccharide takes the place of the O antigen in some of the B. Pertussis population.

LPS damages ciliated tracheal cells by introducing NO (Nitric Oxide) with tracheal

cytotoxin.

Filamentous Hemagglutinin (FHA) and Fimbriae (Fim) are key toxins in B.

Pertussis. These factors aid in the initial step in a Bordetella Pertussis infection, which is

the attachment of the bacteria to the epithelia of the host respiratory tract.

Verified role of adenylate cyclase in pertussis knockout study

Lim, A., et al.4 observes a mutant and non-mutant vaccine tested on mice to determine a possible role of adenylate cyclase. Adenylate cyclase aids in adhesion of the toxin by interacting with epithelial cells in the trachea. A new vaccine was tested with and without adenylate cyclase (CyaA) on mice. The mutant without adenylate cyclase still adhered reasonably well to mammalian cells however, impairment to survive within macrophages existed compared to non-mutant strain. At high doses in vivo the mutant strain was comparable, however at more reasonable stable vaccine doses it was significantly impaired. Impairment included lack of lung colonization ability which is to be expected as CyaA plays a role in adhesion. Findings support a role of adenylate cyclase (CyaA) in BPZE1-mediated protection through cellular mediated immunity.

II. Regulation

Focus on Transcription factor BvgA

Decker, K. B., et al.5 focuses on the regulator BvgA and its role in transcription activation with bacterial RNA polymerase. The transcription of many of the B. Pertussis genes known to be involved as virulence factors are controlled by the activity of BvgS and BvgA. Together, the Bvg proteins form a two-component system that allows varying gene expression depending on changes in extracellular signals from the environment. At the most virulent gene promoters, phosphorylated BvgA binds upstream of the main promoter sequence. The sites occupied have both high and low affinity sites which fill cooperatively. Activation by phosphorylated BvgA is typically handled by a form of class I and II mechanisms, but the virulence genes, fim2 and fim3, are regulated using an unidentifiable RNA polymerase/activator. The fim genes undergo changes in phase because of an extended cytosine (C) sequence within the promoter which is susceptible to slipped-strand mispairing during replication. These unique and error prone regulation systems explain how B. Pertussis, although lacking in the significant genetic diversity of other pathogens, has been widely successful as a human pathogen.

Further BvgA analysis in cyaA gene activation

Byrd, M. S., et al.6 further analyzes the BvgAS regulatory system. Bordetella

Pertussis relies on a two-component regulatory system BvgAS to control expression of distinct phases. In the Bvg(-) phase, the expression of genes required for mobility, are activated and genes encoding virulence factors are not expressed. In the Bvg(+) phase, genes encoding virulence factors are expressed while genes necessary for motility are repressed which is important during the respiratory infection. Essentially, the system waits to move to the appropriate place before activating virulence factors. Using pGFLIP, a fluorescent reporter, they demonstrated that adenylate cyclase (cyaA), considered to be a late stage Bvg(+) phase gene, is activated in B. Pertussis following a switch from Bvg(-) to Bvg(+) phase conditions. The study shows altered activation of cyaA is not due to changes in the cyaA promoter or in the bvgAS alleles of B. Pertussis, but it may actually be species specific.

III. Comparative Genomics

Comparative Genomics

Park, J., et al.7 examines the genome content of B. Pertussis and various subspecies to generate insight into the adaptation of various strands. Previous comparisons of the three sequenced genomes focused on genome degradation with substantial loss of genome content (up to 24%) associated with adaptation to humans of B. Pertussis. Genome spanning single nucleotide polymorphism (SNP) analysis supports a better evaluation of the relationships between the classical Bordetella subspecies, and a closer link between ovine and human B. parapertussis. Comparative analyses of genome content revealed that only 50% of the pan-genome is conserved in all strains, which shows a great deal of diversity among two closely related pathogens. Analyses suggest possible horizontal gene transfer (HGT) events in multiple encoding virulence factors, including the O-antigen and pertussis toxin. Segments of the pertussis

toxin locus and its secretion system locus appear to have been generated from the classical Bordetella subspecies and are changing in different lineages, suggesting functional irregularities among the classical Bordetellae. Observations reveal that multiple mechanisms, such as point mutations, gain or loss of genes, as well as HGTs, contribute to the phenotype diversity of B. Pertussis subspecies.

Summary of Sequenced strains Table:

• Taken from Park, J., et al. (Need permission to use) . (Complex: based on MLST: Multi-

locus sequence typing)

Phylogenetic tree of eleven Bordetellae species with genome-wide SNP sites

Taken from Park, J., et al. (Need permission to use)

Diversity in virulence factor genes heat map:

Taken from Park, J., et al. (Need permission to use) Absence of a certain gene and presence of a pseudogene are highlighted with white and sky blue color with #, respectively. The * indicates the missing nucleotides due to the status of the genome.

Phylogeny of B. Pertussis

Harrington AT, et al8 generates a phylogenetic tree comparison between Bordetella

species.

• Figure shows 16s rRNA dendrogram. Phylogenetic tree of related Bordetellum species. Taken

from Harrington AT, et al (Need permission to use) Bordetella avium is thought to be

strictly an avian pathogen. However, 16S rRNA gene sequencing identified 2 isolates

from 2 humans with respiratory disease as B. avium and a B. avium–like strain.B. avium

and B. avium–like organisms are rare opportunistic human pathogens.

Lipid A Structure Genome Analysis

Novikov, A., et al.9 analyzes the lipid A endotoxin structure of B. Pertussis. The involvement of LPS (Lipopolysaccharides) was first seen in B. Pertussis infection. The relative fatty acid compositions were found to be similar. LPS causes damage to ciliated tracheal cells by an influx of NO (Nitric Oxide) with tracheal cytotoxin. The B. avium LPS, is bird pathogen while Bordetella hinzii is opportunistic in humans, and Bordetella trematum is a human pathogen. Sequence analyses performed showed that the three strains have homologues of acyl-chain modifying enzymes PagL, PagP and LpxO. They also had LgmA, LgmB and LgmC, which are responsible for the glucosamine modification. Mass spectrometry identified a high amount of glucosamine substitution in the phosphate groups of B. avium lipid A. However, this glucosamine modification was absent from B. hinzii and B. trematum. The acylation patterns of the three lipids were similar, but they were different from Bordetella Pertussis and Bordetella parapertussis. They were also found to be close to the lipid A structure of Bordetella bronchiseptica only differing by the amount of hydroxylation in the branched fatty acid.

The major difference distinguishing B. avium lipid A from B. trematum and B.

hinzii was the presence of Glucosamine residues substituting one or both phosphate

groups. Acylation patterns were shown to be similar in all types.

The lgm locus genes form a proposed pathway: LgmA is predicted to be a glycosyl transferase that adds N-acetyl Glucosamine to the carrier lipid C55-P. LgmC is predicted to deacetylate this product. LgmB, is predicted to transfer Glucosamine from C-55P-GlcN to lipid A. A flippase may also be involved in this pathway to transfer C-55-P-GlcN from the cytoplasmic side to the periplasmic side of the membrane.

In B. Pertussis, the lgm locus is part of the Bordetella virulence gene (Bvg).

Genome analysis indicates that the bvgAS regulatory system genes are present in B. avium, B. hinzii and B. trematum, which implies that the lack of Glucosamine modification of lipid A is not due to the absence of the bvgAS genes. It has been shown that B. Pertussis, B. bronchiseptica and B. parapertussis modify their lipid A with Glucosamine, and they have experimentally connected this function to the lgm locus (lgmA, lgmB and lgmC).

The presence of shortchain fatty acids in the C-3 position appears to be a unique

signature for the two human Bordetella pathogens responsible for whooping cough (B.

Pertussis and B. parapertussis.) It is generally accepted that these two pathogens of whooping cough branched from B. bronchiseptica, which expresses a high degree of differentiation at the fatty acid substitution level. The short chain fatty acids at C-3 may be one of the structural characteristics allowing for an advantage to infection of human hosts. Modifications in lipid A structures have been shown to play a role in modulating immune responses to allow bacteria to evade the immune system.

Proteomic Characterization Total Proteins

Williamson, Y. M., et al.10 analyzes proteomic characterization of the total

proteins in B. Pertussis. 163 common proteins were found between the 4 strands

tested.

Table 2A

Strains Total number of proteins

T 182

C 175

D 175

F 176

Strain combinations Total number of common proteins

T,C,D,F 163

T and C only 1

T and D only 3

T and F only 0

C and D only 0

C and F only 0

D and F only 0

T, C and D only 1

T, C and F only 5

T, D and F only 2

C, D and F only 3

Strains Total number of unique proteins

T only 7

C only 2

D only 3

F only 3

Redrawn from Williamson, Y. M., et al. (Need permission to use) This shows summary of the total

number of membrane fraction among the strains assessed in the study. Numerical values are based on a

greater than 95% Scaffold protein id probability. Abbreviations: T = Tohama I, C = C056, D = D946 and F

= F656.

Extended Table with all 163 proteins and some functions may be found from Williamson, Y. M., et al.

(2012). "A gel-free proteomic-based method for the characterization of Bordetella Pertussis clinical

isolates." J Microbiol Methods 90(2): 119-133. (See Table 2D page 124).

Three recent Pertussis circulating isolates predicted to have a role in antibody-

mediated immunity were examined using proteomics to identify any changes in surface

protein expression. The identified proteins included secreted proteins, toxins, outer

membrane proteins which aid in cell membrane synthesis, cell transport, adhesion,

pathogenesis, and/or virulence.

Some common proteins were identified in a combination of two or three strains but were absent in the remaining. Proteins detected only in T while absent in C, D, and F, included CTP synthetase, HP Bp 1123 and, a putative ketopantoate reductase for example.

Proteins were also found exclusive to C, D, and F while not present in T, such as

a putative periplasmic solute-binding protein. Proteins that were identified exclusively to

C, D, or F included HP Bp 3441, a trigger factor, and an exopolyphosphatase.

Subcellular localization of identified proteins. Taken from Williamson, Y. M., et al. (Need

permission to use)

Protein function of identified proteins. Taken from Williamson, Y. M., et al. (Need permission to use)

IV. Clinical Implications Field Studies

Thierry-Carstensen, B., et al11 studies vaccine efficacy in the field with clinical effects. A Monocomponent acellular pertussis (aP) vaccine, from SSI in Denmark has controlled B . Pertussis since 1997. A 71% double-blind clinical trial was conducted for the Denmark vaccine, in 500,000 children it was effective 93% after 3 doses for

infections requiring hospitalization and 78% effective against pertussis not requiring hospitalization.

IgG antibodies (IgG anti-PT) response rates were higher in this monocomponent

aP vaccine with 20μg pertussis toxoid, which was inactivated by hydrogen peroxide 92% without compromising safety. Other two multicomponent aP vaccines were typically inactivated by formaldehyde/glutaraldehyde. A three-component aP had 8μg pertussis toxoid (77.2%) and 5-component aP had 2.5μg pertussis toxoid (47.1%). Denmark solely used this monocomponent aP vaccine for 15 years, and there has been no pertussis epidemic since 2002 (only 36 per 100,000 people). This is in contrast to neighboring countries where epidemics have occurred. The vaccine can be used in combination vaccines for booster vaccinations against pertussis in all ages.

Overview of Symptoms/Treatment strategies for Pertussis

Bentley, J., et al.12 discussed the symptoms and diagnosis of B. Pertussis. Symptoms may be alleviated with the vaccine, however an adult acquiring the disease and passing it on to unvaccinated children is a problem. Toxins in B. Pertussis paralyze cilia causing tissue damage and inflammation which leads to a buildup of mucous. The onset of B. Pertussis infection can predispose the individual to immune-compromised diseases such as pneumonia. The pertussis disease stage lasts 7-8 weeks total.

There are three phases involved in infection. The catarrhal stage lasts 1-2 weeks and results in a dry, unproductive cough, nasal drainage, sore throat, and low fever. The next stage called paroxysmal is a highly contagious stage and can last 1-10 weeks with frequent coughing episodes. Coughing episodes involve coughing without taking a breath until the lungs are empty of air, resulting in the “whoop” which occurs due to partially closed vocal chords. Convalescent stage lasts 2-3 weeks to several months where symptom severity improves although coughing may persist.

Generally, infants may have worse and more prolonged symptoms. Infants

struggle with the disease as they lack the ability to cough due to weakness in their underdeveloped bodies. Symptoms include gagging, gasping for air, choking, and vomiting. In older children these symptoms may be unrecognizable without the whoop, but sweating episodes, feeling faint, or exhaustion from coughing are typical.

Diagnosis is made if the symptoms present are consistent with the disease. The

diagnosis is further supported if the individual was known to be in contact with someone suspected of having whooping cough or infants and children who have not been vaccinated fully. Hospital admission is considered for infants 6 months and younger, as they are unable to cope with the symptoms unvaccinated.

Most people do not develop serious complications, but around 24% of infants

with pertussis can experience related health problems. Minor complications are weight loss, repeated vomiting, and ulceration of the tongue. Severe complications include pneumonia and seizures. Pulmonary hypertension is a serious complication which may lead to cardiac failure and shock. Leukocytosis, with a white blood cell count more than

100,000 is linked to increased fatality among patients. It seems pertussis itself is not particularly deadly but rather the complications that arise such as pneumonia that are responsible for most deaths. Conclusion

Bordetella Pertussis represents a very capable bacterium that can modulate its

response to the host immune system. Further research and testing must be done on

future capable vaccines to ensure that mutation is not present and the proper

vaccinations are conserved for safety and efficacy. Gaining an understanding of the

main virulence factors responsible for the infection in human hosts will allow a greater

range of vaccine capabilities to ensure that the vaccine will have positive results and

maintain stable throughout the years as vaccines must adapt with B. Pertussis and not

against it.

References

1 Fedele, G., et al. (2013). "The virulence factors of Bordetella pertussis: talented modulators of host immune

response." Arch Immunol Ther Exp (Warsz) 61(6): 445-457. 2 Parton, R. (1999). "Review of the biology of Bordetella pertussis." Biologicals 27(2): 71-76.

3 Fedele, G., et al. (2013). "The virulence factors of Bordetella pertussis: talented modulators of host immune

response." Arch Immunol Ther Exp (Warsz) 61(6): 445-457. 4 Lim, A., et al. (2014). "Protective role of adenylate cyclase in the context of a live pertussis vaccine candidate."

Microbes Infect 16(1): 51-60. 5 Decker, K. B., et al. (2012). "The Bordetella pertussis model of exquisite gene control by the global transcription factor

BvgA." Microbiology 158(Pt 7): 1665-1676. 6 Byrd, M. S., et al. (2013). "An improved recombination-based in vivo expression technology-like reporter system

reveals differential cyaA gene activation in Bordetella species." Infect Immun 81(4): 1295-1305. 7 Park, J., et al. (2012). "Comparative genomics of the classical Bordetella subspecies: the evolution and exchange of

virulence-associated diversity amongst closely related pathogens." BMC Genomics 13: 545. 8 Harrington AT, Castellanos JA, Ziedalski TM, Clarridge JE III, Cookson BT. Isolation of Bordetella avium and novel

Bordetella strain from patients with respiratory disease. Emerg Infect Dis [serial on the Internet]. 2009 Jan [date cited].

Available from http://wwwnc.cdc.gov/eid/article/15/1/07-1677.htm 9 Novikov, A., et al. (2013). "Complete Bordetella avium, Bordetella hinzii and Bordetella trematum lipid A

structures and genomic sequence analyses of the loci involved in their modifications." Innate Immun. 10

Williamson, Y. M., et al. (2012). "A gel-free proteomic-based method for the characterization of Bordetella pertussis

clinical isolates." J Microbiol Methods 90(2): 119-133. 11

Thierry-Carstensen, B., et al. (2013). "Experience with monocomponent acellular pertussis combination vaccines for

infants, children, adolescents and adults--a review of safety, immunogenicity, efficacy and effectiveness studies and 15

years of field experience." Vaccine 31(45): 5178-5191. 12

Bentley, J., et al. (2013). "Whooping cough: identification, assessment and management." Nurs Stand 28(11): 50-

57.