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Summary
Outside and inside, pathogen microorganisms manage to live in all of our cell tissues. But what makes them kind of invisible for our sophisticated immune system? By now we know that illness causing microbes somehow must exploit regulatory processes and signal pathways of their hosts. For a detailed understanding of these host-pathogen interactions , investigators now want to uncover how pathogens manage to evade the immune system and how the metabolism of microbes is connected to their hosts. In times of multi-resistant bacteria, a systematic view on the processes involved and high-throughput biotechnology could help to discover urgently needed new drug targets to better fight infectious diseases.
03 March 2011 Martina Bünnige
New Strategies to Fight Infectious Diseases - Arms Race on a Microscale
They live in and on us: bacteria, viruses and other parasitic microorganisms are omnipresent. Our body has numerous effective defense mechanisms at the ready, so that unwanted intruders don’t become a problem in the first place. But pathogenic microorganisms probably have just as many clever tricks up their sleeves to evade their hosts’ immune defense.
Antibiotics provide massive support to eukaryotic organisms as they resist pathogens. These substances impede the growth of bacteria or kill them off. Unfortunately, they also select bacterial strains that show resistance to the chemicals. Is it just a consequence of the improper use of this former panacea? Or a kind of arms race on a microscale?
Modern research, however, no longer views pathogens as mere illness-causing intruders, but as organisms optimally adapted to life in the host. Therefore, studying the biochemical interactions of hosts and infectious agents may be the key to finding novel strategies to fight the diseases that result from them. This work involves answering a number of questions such as how pathogens obtain nourishment and reproduce in their hosts. How do hosts react to unwanted guests? And how do disease agents manage to circumvent immune defenses?
This latter phenomenon, called "immune evasion", is crucial in the development of disease. A detailed understanding of how it occurs will likely be essential in finding the next long-term solution to dangerous infections.
Wolves in Sheep’s Clothing
The complement system is the first barrier that illness-causing germs must overcome upon entering the body. This system involves interactions between more than 40 proteins, phagocytes and antimicrobial peptides, and it provides a quick and effective response to most intruders. It is a part of the innate immune response and was neglected for a long time in research, overshadowed by the apparently more sophisticated adaptive immune system. Today we know that the complement system is crucial in terms of bacteria's immune evasion, and a better understanding of its biology may be useful in the design of novel therapies.
The proteins of the complement system circulate through the blood plasma and ward off invaders, sparing only cells that have the right “identity card”. Normally the immune system doesn't attack healthy tissues because they are protected by molecules on their surfaces or which circulate in the plasma. A key player in this system is the protein factor H. It helps distinguish "friend from foe" by administering a test – it binds to human molecules, but not those of foreign origin. Some microorganisms cheat by trapping factor H on their surfaces with the help of "CRAS" proteins ("complement regulator-acquiring surface proteins"). Like wolves dressed in sheep’s clothing, this allows them to slip past the complement system without being recognized.
Understanding this bacterial masquerade may provide a way to defeat it. CRAS proteins lie on the surface of the microbe, which puts it into contact with proteins in serum. This might also permit them to be recognized by antibodies, making them a potential target for the development of a vaccine. This approach is the subject of ongoing clinical trials in which a vaccine targets the factor H-binding protein of Neisseria meningitidis, a bacterium that causes rare, but often deadly, forms of meningitis and sepsis.
Molecular Hide and Seek
Pathogens that cannot evade the complement reaction are normally digested by white blood cells called phagocytes; once inside the cell, they are delivered to a compartment called a vacuole. It fuses to other compartments called lysosomes, which bear enzymes that degrade molecules, and the invaders are usually destroyed. But even some bacteria that have been trapped in vacuoles have evolved strategies to escape destruction. Salmonella enterica manages to survive and replicate in the compartment, through a clever strategy in which it builds a sort of hypodermic needle made of proteins. It uses the needle to discharge "effector proteins" into the cell cytoplasm, and they suppress the host’s immune response.
Other invasive microorganisms manage "jail-breaks" from the vacuole. Listeria, for example, secrete proteins that build openings in the vacuole’s membrane. The bacteria use them to escape into the cytoplasm, where they can multiply and eventually infect neighboring cells. The trick here is that the host itself helps the bacteria to escape unwittingly. A factor called GILT ( γ -interferon-inducible lysosomal thioreductase), produced by the cell, plays an important role in antigen processing. Here, it is kind of high jacked by Listeria in order to increase the activity of it own pore-forming protein. Knockout mice that don’t express GILT are safe from infections with Listeria.
The effects of bacteria on our health are as diverse as their strategies to evade the host’s immune defenses. Our understanding of these multifarious and highly complex molecular interactions have made great strides in recent years thanks to tremendous advances in biotechnology.
Intelligently Searching a Haystack
Today's researchers have on hand a wide array of high-throughput methods that allow them to sequence proteins and genomes, analyze chemical structures, and use biochips to analyze the cell's population of RNA and protein molecules. These technologies produce masses of data that have to be analyzed using bioinformatic methods. As a result, scientists have systematic techniques to search for the needle in the haystack, in this case promising targets for drugs.
One center that produces, collects and collates data from high-throughput technologies is the National Institute for Allergy and Infectious Diseases (NIAID) Biodefense Proteomics Program in the USA. NIAD's Proteome Research Centers focus on proteins because they are central actors in the biochemical interactions of hosts and agents.
By screening protein-protein interactions (PPI), the US researchers have, for example, identified more than 3000 interactions between 1700 human proteins and 943 Bacillus-anthracis proteins. Many of the molecules that turned up are well-known actors in human immune defenses – such as the proteins that form the major histocompatibility complex (MHC). MHC molecules play a central role in immunity, because they bind to foreign molecules that have been digested by the cell, then carry them to the surface where they can be detected by other components of the immune system.
Here, too, bacteria have evolved ways of interfering with the host's defenses. Yersinia pestis produces proteins that interfere with the behavior of a human molecule called NFkB, which activates genes to stimulate a range of immune and inflammatory reactions. NFkB is so potent that it has to be carefully controlled; if its behavior is blocked, the T cells that normally display foreign molecules to other cells do not develop properly, and they fail to trigger a wider immune response. Y. pestis has proteins that affects NFkB in two ways: they directly inactivate it, and they also block molecules that normally prevent this from happening. Therefore Y. pestis attacks not only the guard itself, but also the guard’s guard, and can colonize the host cell without being recognized and destroyed.
Cutting off Supply Channels
„Once a host is infected, life is free and easy“, could be the motto of a microbe that has managed to circumvent human immune defenses. High-throughput technologies are also being used to study interactions between pathogens and their hosts in a priority program of the German Research Foundation (DFG). Launched in 2008 with the name „Host-adapted Metabolism of Bacterial Infectious Agents“, the project draws together infection biologists, physicians, metabolism physiologists, bioanalysts and bioinformaticians.
This title derives from the fact that bacteria and other parasites help themselves to the rich offerings of their host's biology, for example by tapping into the cell's metabolism to nourish themselves. The researchers now want to find out exactly how they manage this feat. Cutting off the supply channels exploited by the microbes could suggest new approaches for therapies.
“Fluxomics” is the trendy new word that describes a clever, automatic technique to measure metabolism flows under complex conditions in infected cells. Bacteria are fed the stable carbon isotope 13C, in the form of glucose or another substance. When the carbon-containing molecule is broken down, researchers use methods such as NMR or GC-MC to measure and quantify the accumulation of 13C. By studying its accumulation in the amino acid alanine, for example, researchers can measure how efficiently the cell is taking up glucose and converting it to alanine. This creates an "isotopologue profile" that changes over time and sheds new light on biochemical pathways within the microbes.
One discovery has been that when pathogens infect cells, they use different metabolic pathways than when grown in culture. The researchers also found that a fundamental metabolic process by which cells derive energy, called the citric acid cycle, is not closed with Listeria. One step, in which oxaloacetic acid is converted to pyruvic acid, requires a new enzyme. This essential reaction requires a special carboxylase, which might serve as a target for novel antibiotic pharmaceutical substances.
Always a Step Ahead
Meanwhile, the arms race continues. The practice of broad-spectrum antibiotics, by which a few substances are used to kill many types of pathogens, has been proven to be unsustainable. It has frequently led pathogens to develop multi-drug resistance, and researchers are struggling to stay a step ahead of the bacteria's rapidly evolving survival strategies.
What researchers are learning about the interplay of pathogens and their hosts is also permitting them to use new combinations of molecules as a way of strengthening existing antibiotics. In response to chemical attacks, for example, bacteria have developed beta-lactamases that reduce the effects of penicillin, cephalosporin and carbapenem. Adding a substance that blocks the beta-lactamases can restore their potency, but for how long? Cases are already known in which bacteria have developed resistance to the inhibitors, too.
Another promising approach is to slip iron (which is essential for bacterial growth) into the cell in the form of a "Trojan horse". Researchers managed to attach highly potent antibiotics to synthetic molecules called siderophores, which act as transport vehicles for the iron. Due to the siderophore-mediated iron uptake system, this can kill off even bacteria that have developed resistance to multiple drugs because of high concentrations in the bacteria's cytosol.
Current treatments for infectious diseases aim to kill invaders, but more creative solutions are being considered. It might be possible to develop therapies in which infectious agents are permitted to settle permanently in their hosts instead. If so, the microbes wouldn't be forced to evolve to survive, and would likely develop resistance at a much slower pace (if at all). But this approach will depend on obtaining a global view of humans and microbes using "systems biology" methods that are still in their infancy. While these new tools have not yet produced a change in the way diseases are treated on a daily basis in the clinic, they have already yielded deep insights into the interplay between hosts and infectious agents.
References and additional reading:
Briken V (2008) Molecular Mechanisms of Host-Pathogen Interactions and their Potential for the Discovery of New Drug Targets. Curr Drug Targets 9 (2): 150-157. (Review, free full text)
Collier RJ. (2010) Microbiology. Salmonella's safety catch. Science 328(5981):981-2. doi:10.1126/science.1190758
Comas I, Gagneux S (2009) The Past and Future of Tuberculosis Research. PLoS Pathog 5(10): e1000600. doi:10.1371/journal.ppat.1000600. (Review)
Dyer MD, Neff C, Dufford M, Rivera CG, Shattuck D (2010) The Human-Bacterial Pathogen Protein Interaction Networks of Bacillus anthracis, Francisella tularensis,and Yersinia pestis. PLoS ONE 5(8): e12089. doi:10.1371/journal.pone.0012089.
Eisenreich W, Dandekar T, Heesemann J, Goebel W (2010) Carbon metabolism of intracellular bacterial pathogens and possible links to virulence. Nat Rev Microbiol 8(6):401-12. doi:10.1038/nrmicro2351
Jiang HQ, Hoiseth SK, Harris SL, McNeil LK, Zhu D, Tan C, Scott AA, Alexander K, Mason K, Miller L, DaSilva I, Mack M, Zhao XJ, Pride MW, Andrew L, Murphy E, Hagen M, French R, Arora A, Jones TR, Jansen KU, Zlotnick GW, Anderson AS (2010) Broad vaccine coverage predicted for a bivalent recombinant factor H binding protein based vaccine to prevent serogroup B meningococcal disease. Vaccine. 2010 Aug 23;28(37):6086-93. doi:10.1016/j.vaccine.2010.06.083
Laarman A, Milder F, van Strijp J (2010) Complement inhibition by gram-positive pathogens: molecular mechanisms and therapeutic implications. J Mol Med 88:115-120. doi:10.1007/s00109-009-0572-y
McGarvey PB, Huang H, Mazumder R, Zhang J, Chen Y, Zhang C, Cammer S, Will R, Odle M, Sobral B, Moore M, Wu CH (2009) Systems Integration of Biodefense Omics Data for Analysis of Pathogen-Host Interactions and Identification of Potential Targets. PLoS ONE 4(9): e7162. doi:10.1371/journal.pone.0007162
Moellering Jr. RC (2011) Discovering new antimicrobial agents. Intern J Antimicr Agents 37: 2-9. (Review, abstract)
Schär J, Stoll R, Schauer K, Loeffler DI, Eylert E, Joseph B, Eisenreich W, Fuchs TM, Goebel W (2010) Pyruvate carboxylase plays a crucial role in carbon metabolism of extra- and intracellularly replicating Listeria monocytogenes. J Bacteriol 192(7): 1774–1784. doi:10.1128/JB.01132-09
Singh R, Jamieson A, Cresswell P (2008): GILT is a critical host factor for Listeria monocytogenes infection. Nature 455, 1244-1247 (30 October 2008) | doi:10.1038. doi:10.1038/nature07344