Pigs, Pathogens, and Possible Progressive Pharmaceuticals?

Original Article:  Wang, A., L. Yi, S. Wang, H. Fan, C. Ding, X. Mao, and C. Lu. (2015). Crystal Structure and Identification of Two Key Amino Acids Involved in AI-2 Production and Biofilm Formation in Streptococcus suis LuxS. PLoS ONE10(10): e0138826

Post By: Kimiko Wysocki

 Source

The first thing that probably comes to mind while observing the piglet above is its overwhelming cuteness. One thing you’re probably not thinking about is Streptococcus suis, a bacteria and important pathogen of pigs.

What is Streptococcus suis?

S. suis is zoonotic pathogen from swine, responsible for major economic loss is the porcine industry.  A zoonotic disease is a disease that can be passed from animals to humans, caused by viruses, bacteria, parasites and fungi.  Thus, S. suis is the causative agent of serious infections in pigs and in humans who have close contact with pigs.  The pathogen causes symptoms of meningitis and septicemia, sometimes leads to death.  S. suis infections have been shown to be the primary cause of meningitis in adults in Vietnam and have caused the death of 39 people in China from a single outbreak. So what makes S. suis infections so severe? Biofilms!

This is a graph of the annual proportion of patients admitted to the Hospital for Tropical Diseases in Ho Chi Minh City, Vietnam for suspected bacterial meningitis. The gray represents patients infected with S. suis , the black bar represents other bacterial infections, while the dashed bar are unconfirmed bacterial meningitis. Source

This is a world map of S. suis cases with background pig density data. Source

What are Biofilms?

Biofilms are multicellular aggregates of bacteria held together by an extracellular polysaccharide (EPS).  The EPS provides a diffusion barrier, protecting the bacteria against antibiotics, predators, and immune cells. The high cell density in the biofilm also allows for a number of processes, one of which is quorum sensing. Quorum sensing is a form of cell-to-cell communication that bacteria use to monitor their own population density through production and exchange of signal molecules. An example of this is LuxS. LuxS is a metabolic enzyme that plays a key role in the production of the signal molecule AI-2. AI-2 can mediate communication between species of bacteria in order to regulate bacterial behavior to form a biofilm. Therefore, since LuxS is responsible for bacterial virulence factors (properties of the bacterium that contribute to pathogenicity), authors Wang et al. have investigated the potential of S. suis LuxS as an antibiotic target. Source

The Study

Wang et al. chose to study LuxS from S. suis because it is evolutionarily conserved.  However, many recent studies have discovered the presence of LuxS in a variety of bacteria.  In order to determine the crystal structure of LuxS S.suis and to form a comparison between LuxS and other species, the authors used size exclusion chromatography. Wang et al. found that LuxS S. suis is a monomer made of four anti-parallel beta sheets and four anti-parallel alpha helices.  When comparing the crystal structure of S. suis LuxS to the structure of other bacterial species, they found an overall structural conservation.

The overall structure of S. suis LuxS, four 

LuxS monomers arranged asymmetrically.

 Each color represents a different monomer. 

A ribbon showing an overall comparison

 of the structure of S. suis LuxS against 

four other bacteria. Yellow represents

Haemophilus influenza, green represents

Deinococcus radiodurans, cyan

 represents Helicobacter subtilis, 

and grey represents Bacillus subtilis. 

 A bioinformatics analysis of amino acid sequence alignment showed that S. suis LuxS shares 22.5-68% sequence identity with LuxS proteins from other bacterial species. The analysis also showed the two important residues in LuxS proteins for catalytic reactions (Met-80 and Tyr-87) were replaced in S. suis LuxS protein to Phe80 and His87 respectively.

  

In order to figure out the roles of Phe80 and His87 in catalysis and substrate binding, Wang et al. mutated Met and Tyr to F80M and H87Y respectively.  These mutated forms of the protein were expressed and the authors studied their catalytic parameters in comparison to a wild type protein as well as a control mutation of Cys82 to C82A.  The control mutation acted as expected and lost all catalytic activity. However, the F80M mutant had a 7-fold decrease in activity compared to the wild type while the H87Y mutant had a 37-fold decrease in activity. A mutation of both F80M and H87Y led to an overall decrease in activity that was 42 times less that then wild type protein.

Determination of biofilm formation of the mutated LuxS and wild type LuxS

Next, based on these observations, the authors mutated Phe-80 and His-87 to a methionine and tyrosine to investigate the functional importance of F80M, H87Y, and the double mutation in AI-2 production. This was carried out in the plasmid pSET-luxS.  This yielded 54%, 75%, and 41% of AI-2 production in F80M, H87Y, and the double mutation respectively in comparison to the parental strain.  The authors deduced that this means that F80 and H87 mutations affect the production of AI-2.

Determination of biofilm formation of the

 mutated LuxS and wild type LuxS

Lastly, Wang et al. analyzed the biofilm formation between parental and mutant strains using a 96 microtiter plate assay. This showed that the F80M, H87M, and double mutation had slightly decreased biofilm formation than the parental strain.

In Conclusion…

In conclusion, the research by Wang et al. showed that S. suis LuxS is structurally similar to the LuxS protein in other bacteria. However, they found that S. suis LuxS has two important residues that differ from the other bacterial species tested, Phe80 and His87.  Through their experimentation, Wang et al. found that Phe80 and His87 are integral towards the kinetics of the enzymatic reaction of the production of AI-2.  Based on their data the authors suggest that based on the structure of S. suis, the Phe80 and His87 are responsible for substrate binding.  Therefore, the substitution of Phe80 or His87 led to a significant decrease in enzymatic activity and consequently a reduction in AI-2 formation and the ability to form enzymes.  The authors speculated that with more research on the effect of the mutated catalytic residues new antimicrobial drugs can be created that target the amino acids in LuxS to reduce AI-2 production. This in turn will lead to the decrease in biofilm formation and the decrease of virulence of pathogens such as S. suis. However, despite its promises towards new antimicrobial drugs, S. suis is a widely neglected bacterium in the realm of microbiology research.  Recently, Willenborg et al. have been studying the role of transcriptional regulators and their involvement in virulence of S. suis. Overall, more research investigating the possibility of pharmaceuticals from the pig pathogen S. suis would be greatly beneficial.

References Sited

Wang, A., L. Yi, S. Wang, H. Fan, C. Ding, X. Mao, and C. Lu. (2015). Crystal Structure and Identification of Two Key Amino Acids Involved in AI-2 Production and Biofilm Formation in Streptococcus suis LuxS. PLoS ONE10(10): e0138826

Willenborg, J., A. de Greeff, M. Jarek, P. Valentine-Weigand, and R. Goethe. (2014). The CcpA regulon of Streptococcus suis reveals novel insights into the regulation of the streptococcal central carbon metabolism by binding of CcpA to two distinct binding motifs. Microbiology 157:1823-1833.

http://www.cdc.gov/onehealth/zoonotic