A Genome-Level Search for Bacterial Genes on Which
Positive Selection May Operate: A Means for
Identifying Possible Virulence Factors?

Junaid Gamieldien
junaid@sanbi.ac.za

Winston Hidewinhide@sanbi.ac.za

South African National Bioinformatics Institute, University of the Western Cape
Private Bag X17, Bellville, 7535, South Africa

1 Introduction
Positive selection is very important for the evolution of a new or improved protein function, as itincreases the probability that an advantageous mutation becomes established in a population. Previousstudies have shown that genes on which positive selection may operate can be identi ed by comparing
synonymous substitution rates (KS ) and nonsynonymous substitution rates (KA ), where KA > KS .
It has been previously shown that positive selection may operate on the virulence genes of pathogens
to enable them to maintain or improve their `advantage' over their hosts. KA =KS analysis of genes
coding for cell surface proteins of the malaria parasite, Plasmodium spp., have identi ed gene regionsthat have nonsynonymous substitution rates that are signi cantly higher than the synonymous rate[3]. Since host antibodies target these protein regions, it has been suggested that this phenomenon isevidence of positive selection-based diversi cation of the protein to maintain an advantage over thehost immune system. A similar phenomenon has been demonstrated in highly pathogenic HIV1 versus
mildly pathogenic HIV2 and in highly pathogenic versus mildly pathogenic SIV [4]. A broader study
by Endo et al. [2] analyzed approximately 3600 groups of homologous sequences obtained from DNA
sequence databases and found 17 candidate groups that showed evidence for positive selection. Ofthese, 9 groups were the surface antigens of parasites or viruses.
A number of bacterial virulence-associated genes have been demonstrated to be under positiveselection. Recently, the cytotoxin-associated gene A (cagA) of Helicobacter pylori, found in nearly
all isolates from patients with peptic ulcer disease, has been shown to be under positive selection[5]. It has also been suggested that two proteins from pathogenic Neisseria species and Haemophilus
species, which play a role in iron acquisition from host transferrin, are under positive selection [1].
This indicates that positive selection may operate on pathogen virulence genes other than those coding
for surface antigens.
We are developing a system for the identi cation of bacterial virulence genes at a raw, wholegenome level, using KA =KS ratios. Here, we present a brief introduction to the system and ourpreliminary results.

2 Methods
To test our hypothesis, we have performed two intraspecies KA =KS comparisons using genomic sequences from the di erent strains of Mycobacterium tuberculosis and Neisseria meningitidis, available
from the Sanger Center and The Institute for Genomic Research.

269The major steps in the method are:a. Open reading frame prediction
b. Searching for identical genes in each intraspecies ORF set
c. DNA alignment (built from protein alignments)
d. Windowed calculation of KS and KA
e. Selection of genes that have regions where KA > KS
f. Sequence similarity searches of candidates for their functional annotation

3 Results
For M. tuberculosis, we have identi ed a number of genes under positive selection that may biologically be associated with virulence. A large percentage of these genes code for enzymes that produce
modi ed fatty acids found uniquely in the cell walls of pathogenic mycobacteria. Others include atranscription regulator of virulence factors, transmembrane proteins, and a protein that has a potential role in adaptation of the pathogen to intracellular (macrophage) conditions. Analysis of thelimited N. meningitidis genomic sequences produced similar results. These included genes coding fora hemoglobin receptor, which plays a role in acquisition of host iron; two host-cell adhesion proteins;and an enzyme involved in serum resistance of the pathogen. A number of genes were previously
reported to code for virulence factors.

4 SummarySince a number genes detected were previously reported to be virulence genes, and many other candidates may be associated with virulence based on their predicted function, there is suggestive evidence
that the system has the potential to identify many virulence genes from the raw genomic data of abacterial pathogen.

References[1] Cornelissen, C.N. and Sparling, P.F., Iron piracy: acquisition of transferrin-bound iron by bacterial pathogens, Mol. Microbiol., 14(5):843{850, 1994.
[2] Endo, T., Ikeo, K., and Gojobori, T., Large-scale search for genes on which positive selection
may operate, Mol. Biol. Evol., 13(5):685{690, 1996.
[3] Ohta, T., Cirumsporozoite protein genes of malaria parasites (Plasmodium spp.): evidence forpositive selection on immunogenic regions, Genetics, 127(2):345{353, 1991.
[4] Shpaer, E.G., Mullins, J.I., Rates of amino acid change in the envelope protein correlate withpathogenicity of primate lentiviruses, J. Mol. Evol., 37(1):57{65, 1993.
[5] Van der Ende, A., Pan, Z., Bart, A., Van der Hulst, R.W.M., Feller, L., Xiao, S., Tytgat, G.N.J.,
and Dankert, J., cagA-Positive Helicobacter pylori populations in China and The Netherlands
Are Distinct, Infect Immun, 66(5): 1822{1826, 1998.

270