Humans Vs Superbugs: The Combat Strategy!

Story until now in ‘Humans Vs Superbugs’ series: Breakthrough discovery of 1st antibiotic penicillin opened the floodgate of sort for development of plethora of anti-microbial, giving humans advantage over microbial infection by saving lives over the wide range of microbial infection. However, it didn’t take much time for these tiny smart cellular machinery to strike back through AMR microbes with capability to send our civilization back to Stone Age (little exaggeration!). Off course, human complacency and laxity like antibiotic abuse and lack of chemical innovation, played significant role, as explained in last post.

Story ahead…..

The marvelous survival mechanisms of pathogenic microbes require an equally effective counter response from humans. An important front in the campaign would be to diversify the already established traditional cellular targets, viz. cell wall synthesis, protein synthesis, ribosomal inhibitors, etc. To a more intricate drug-able targets, which are not necessarily important for microbial survival but play a vital role in pathogenic virulence (ability of the organism to invade the tissue of host). In this particular context, one of the recent scientific communication in 'Journal of American Chemical Society. 2014, 136, 1579?1586' has put forward an interesting concept regarding metal acquisition requirement in host for any pathogenic microbe.

Trace metals like iron is required by almost all living organisms for their routine metabolism (chemical processes that occur within the cells or tissues, for regular synthesis or breakdown of useful building blocks by living organisms). The importance of free iron can be estimated from the fact that its availability in mammalian host is almost negligible for survival of any invading bacteria. Iron in host is usually found in tightly bound form with transport macromolecules like transferrin and lactoferrin in various secretory fluids. In order to circumvent the scarcity of free nutrients in host system, pathogenic microbes have devised an incredible mechanism. Whereby, they synthesize virulence associated 'siderophores (metal scavengers)' and release them to compete with host sequestration system to efficiently scavenge iron and return it to microbial cell

Isn’t this astonishingly smart? It’s a remarkable mechanism of deploying physiologically superior guerrilla fighters to systematically snatch ration from an invaded territory, to gain a self-sustainable ground in a fierce dispute.

Exactly, this is where the opportunity lies, if biosynthesis of siderophores could be eradicated in order to restrict important nutrient acquisition; that can starve pathogens to death without exerting selective pressure for developing resistance. To lay out the strategy neatly, any macromolecule (like siderophores) are synthesized by the producing organism (in this case staphyloferrin B from Staphylococcus aureus) in a sequential enzymatic fashion where different functional proteins assemble individual portion of the molecule to build an active chemical architecture. Therefore, it was envisioned to develop an antibiotic to specifically target one of the assembly line protein for disruption of whole siderophore making machinery and hence starving invading microbes to death.

Booooom……. here you go bad a**! 

The disruption of microbial arsenal was meticulously strategized based on inhibition of the earliest enzyme (SbnE) in the assembly line. To achieve same, sbnE gene was cloned into an E.coli vector and was expressed to obtain sufficient protein for development of an in vitro assay (method to study processes in an artificial environment like test tube, outside the living organism). 

It is like doing a mock military exercise in a sophisticatedly designed simulation for an impending war situation.

The biochemical assay was fastidiously designed based on the native function of enzyme SbnE to catalyze an ATP dependent condensation reaction, producing diphosphates in process. Diphosphates were then cleaved by externally providing inorganic pyrophsphatase (PPi) enzyme to make free phosphates which when encounters malachite green reagent, emits bright green color and can be observed using spectrophotometer (sometime by trained naked eyes as well). The idea behind the robust in vitro assay was incredibly simple, if the enzymatic reaction gets incubated with an inhibitor of SbnE, then diphosphates will not be produced and therefore no bright green color, or vice versa. Now, next objective was to find an effective inhibitor of the SbnE enzyme towards a greater cause of identifying new antibiotic.

Now, that there is a seemingly fantastic sabotage plan against enemy, but recruits are still at large and without the properly equipped recruit, the meticulously planned commando operation is a distant dream.

So, it turned out, we may be able to beat these superbugs and that too playing by their rules itself. But the question still looms, where are the effective potentially novel molecules for the purpose? If they exist, how to identify them? If they don’t than what next?

WELL.... HOPE TO FIND THE ELUSIVE MOLECULES TO KNOCK OUT THIS SMART TINY DUDES IN NEXT AND LAST POST OF THIS SERIES!