Dear germ
Since the time of Pasteur, it has been known that the human gastrointestinal tract is essentially a flow-type bioreactor in which many microorganisms live. The attitude of scientists to the intestinal microflora has changed radically during this time. A hundred years ago, the great Ilya Mechnikov, the founder of the modern theory of immunity, for the creation of which he received the Nobel Prize (for two with his irreconcilable opponent, the equally great Paul Ehrlich), even proposed the removal of the large intestine as one of the ways to prolong life. And for those to whom this measure seemed too radical, he recommended drinking as much kefir as possible in order to displace harmful, in his opinion, microbes with beneficial lactobacilli. After half a century, the course changed by 180 degrees. It turned out that the normal intestinal microflora, as well as the skin and mucous membranes, performs many useful functions – for example, suppresses the vital activity of pathogenic microorganisms constantly attacking the body. And in recent years, the bravest of microbiologists have gone even further, declaring man and his microbes a single symbiotic superorganism.
The development of methods of molecular biology has brought scientists to a new level of understanding of the processes of symbiosis of man and his microflora, which seemed well studied and from the study of which no special surprises were expected. The rapid growth in the speed and falling cost of DNA sequencing methods (determining its nucleotide sequence) and the parallel growth in the power of personal computers and the development of the Internet made it possible to analyze information about large sections of genomes. After the chromosomes of hundreds of species of individual bacteria were deciphered, a new approach appeared in the genetics of microorganisms – a population approach: the analysis of the genes of all bacteria inhabiting a certain area at once. Of course, the population of the "human bioreactor" turned out to be one of the most important for the study of microbial populations.
The first work that forced a new look at the intestinal microbiota was published in 1999 by a group of scientists from the National Institute of Agronomic Research (France) and the University of Reading (Great Britain). The authors decided to use the 16S RNA gene sequencing method to study the intestinal microbial population.
16S RNA – identification of bacteria Since the time of Pasteur, the first and necessary step in determining microorganisms has been their cultivation on nutrient media.
But many important (and useful, and pathogenic) microbes do not want to grow on any of the media. It became possible to study previously inaccessible uncultivated bacteria and begin to restore order in the extremely confusing systematics of already known prokaryotes with the development of bioinformatics and the advent of modern methods of molecular biology – polymerase chain reaction (PCR), which allows millions and billions of exact copies to be obtained from one DNA site, cloning of genes isolated using PCR in bacterial plasmids and sequencing techniques the nucleotides obtained as a result of all this in sufficient quantity for analysis.
An ideal marker for identifying microorganisms turned out to be a gene encoding 16S ribosomal RNA (each of the two subunits of ribosomes – cellular workshops for protein synthesis – consists of intertwined protein molecules and chains of ribonucleic acids).
This gene is present in the genome of all known bacteria and archaea, but it is absent in eukaryotes and viruses, and if you find a sequence of nucleotides characteristic of it, you are definitely dealing with prokaryotic genes. (To be very precise, the 16S RNA gene is also present in eukaryotes, but not in nuclear chromosomes, but in mitochondrial ones. This once again confirms that mitochondria are distant descendants of symbiont bacteria of the first eukaryotic organisms.)
This gene has both conservative sites, the same in all prokaryotes, and species-specific. Conservative sites serve for the first stage of the polymerase chain reaction – the attachment of the studied DNA to primers (seed sites of DNA to which the studied chain of nucleotides must join to begin the analysis of the rest of the sequence), and species–specific ones - to determine the species. In addition, the degree of similarity of species-specific sites very well reflects the evolutionary relationship of different species.
An additional bonus is that ribosomal RNA itself can be used for cloning and subsequent analysis, which is present in much larger quantities in any cell than the corresponding gene. Only it is necessary first to "rewrite" it in DNA with the help of a special enzyme – reverse transcriptase.
Nucleotide sequences of 16S RNA of all known bacteria and archaea (about 10,000 species) are publicly available. The identified sequences are compared with those available in databases and accurately identify the type of bacterium or declare it to belong to another uncultivated species.
Recently, there has been an intensive revision of the old, phenotypic, classification of bacteria based on poorly formalized criteria – from the appearance of colonies to food preferences and the ability to be stained with different dyes. The new taxonomy is based on molecular criteria (16S RNA) and only partially repeats the phenotypic one.
Coding sequences of 16S RNA were extracted directly from the "environment" using PCR – 125 milligrams of human, sorry, stool, embedded in the plasmids of E. coli (not because it is intestinal, but because Escherichia coli is one of the favorite workhorses of molecular biologists) and isolated again from the culture of multiplying bacteria. Thus, a library of 16S RNA genes of all microorganisms in the sample was created. After that, 284 clones were randomly selected and sequenced. It turned out that only 24% of the obtained 16S RNA sequences belonged to previously known microorganisms. Three-quarters of the microflora found in the intestines of every person has avoided the attention of researchers armed with the methods of classical microbiology for more than a hundred years! Scientists simply could not find the conditions for the cultivation of these bacteria, because the most capricious inhabitants of the intestine refused to grow on traditional microbiological media.
To date, using molecular methods, it has been established that 10 out of 70 large bacterial taxa are represented in the microbiota of an adult. About 90% of our microbes belong to the Firmicutes types (these include, for example, the well–known lactobacilli - the main "culprits" of milk souring) and Bacteroidetes – obligate anaerobes (organisms capable of living only in the absence of oxygen), which are often used as an indicator of pollution of natural waters by sewage. The remaining 10% of the population is divided between the taxa Proteobacteria (these include, among others, E. coli), Actinobacteria (the antibiotic streptomycin was isolated from one of the species of actinomycetes), Fusobacteria (common inhabitants of the oral cavity and a frequent cause of periodontal disease), Verrucomicrobia (a species of these microbes feeding on methane was recently discovered in a geothermal source of which there are plenty in the intestines due to the vital activity of other microorganisms), Cyanobacteria (they are still often called by the old name "blue-green algae"), Spirochaeates (fortunately, not pale), Synergistes and VadinBE97 (what kind of animals are these, ask the creators of the new systematics of prokaryotes).
Despite the fact that the species composition of intestinal microorganisms is quite monotonous, the quantitative ratio of representatives of certain systematic groups in the microbiota of different people can vary greatly. But what is the normal intestinal microflora and what are the ways of its formation?
This question was answered in a 2007 paper published by a group of American biologists led by Patrick Brown from Stanford University. They traced the formation of microbiota in 14 newborn infants during the first year of life. The authors managed to identify several sources of colonization of the gastrointestinal tract. The microbiota of infants had similarities with the mother's microflora: vaginal, fecal, or with the microflora of breast milk samples. Depending on the sources of colonization, different species prevailed in the intestinal microflora of infants during the first year of life. These differences remained significant throughout the entire study period, however, by the age of one year, the features of the formation of an adult microbiota became noticeable. Interesting data were obtained on the example of a pair of twins. Their microflora was almost identical in composition and also changed in the same way. This finding revealed the huge role of the human component of the microbiota-host pair in the formation of the intestinal microflora population. For the purity of the experiment, of course, it would be necessary to separate the babies in the maternity hospital – a wonderful plot for an Indian film! Years later, they recognize each other by analysis… But data from other studies have confirmed the assumption that individual, including hereditary, features of human biochemistry have a great influence on the composition of his microbiota.
There is more microbial in us than human In addition to studying individual types of intestinal microflora, in recent years many researchers have been studying the bacterial metagenome – a set of genes of all microorganisms in a sample of the contents of the human intestine (or in a wash from the skin, or in a sample of silt from the seabed).
To do this, the most automated, computerized and high–performance DNA sequencing technologies are used, which make it possible to analyze short sequences of nucleotides, assemble a puzzle using several matching "letters" at the ends of these sections, repeat this procedure many times for each piece of the genome and get the decoding of individual genes and chromosomes at a speed of up to 14 million nucleotides per hour - orders of magnitude faster than it was done just a few years ago. So it was found that the microbiota of the human intestine has about 100 trillion bacterial cells – about 10 times more than the total number of cells of the host body. The set of genes that make up the bacterial metagenome is about 100 times larger than the set of genes of the human body. If we talk about the volume of biochemical reactions occurring within the microbial population, it is again many times higher than that in the human body. The bacterial "reactor" implements metabolic chains in the host's body that the host is not able to support itself – for example, the synthesis of vitamins and their precursors, the decomposition of certain toxins, the decomposition of cellulose to digestible polysaccharides (in ruminants), etc.
Studies conducted in the laboratory of Jeffrey Gordon (Washington University School of Medicine, St. Louis, Missouri) have made it possible to link the species diversity of bacteria in the gastrointestinal tract with the diet and metabolic characteristics of an individual. The results of the experiment were published in the December 2006 issue of Nature. A year-long experiment was supposed to establish a correlation between a person's excess weight and the composition of the microbial population of his intestines. A dozen fat men who agreed to put their bellies on the altar of science were divided into two groups. One went on a low–fat diet, the second - with a low carbohydrate content. All the volunteers lost weight, and at the same time the ratio of the two main groups of intestinal microorganisms changed: the number of Firmicutes cells decreased, and the number of Bacteroidetes, on the contrary, increased. On a low–fat diet, this change became noticeable later – after patients lost 6% of their weight, and on a low-carb diet - after losing the first kilograms (2% of the initial body weight). At the same time, the change in the composition of the microflora was the more pronounced, the less the weight of the participants in the experiment became.
In parallel, experiments were conducted in the same laboratory on laboratory mice carrying a mutation in the leptin gene – the "satiety hormone", a protein that is synthesized in adipose tissue cells and contributes to the formation of a feeling of satiety. Mice that have both copies of this gene damaged (this mutation is indicated by the Lep ob index) eat 70% more than the wild type, with all the consequences that follow from this. And the content of Firmicutes in their intestines is one and a half times higher than that of heterozygous lines with only one defective allele (ob/+), and homozygous wild-type lines with a normal gene (+/+).
The researchers tested the effect of microflora on the metabolism of its "host" on another model – gnotobiotic mice.
Such animals, who have been living in sterile chambers since birth and have never encountered a single microbe in their lives, are not often used in biomedical research. Absolute sterility in a mouse house, a rabbit house, and even more so in a goat barn is an expensive and troublesome business, and after meeting with the first microbe or virus, the poor fellows will either die or become unfit for further experiments. What happens in gnotobiots with the immune system is a separate story, but they eat for three and at the same time – skin and bones due to the lack of a microbial component of digestion.
After transplantation of microflora from obese (ob/ob) donors, gnotobiotic mice gained almost one and a half times (by 47%) in two weeks. Those who were "seeded" with microflora from wild-type (+/+) donors with normal weight gained only 27%.
The results of further study of changes in the symbiotic mouse-microbial organism brilliantly confirmed the hypothesis that the microbiota of obese individuals contributes to deeper food processing. Comparison of stool DNA samples from obese and normal mice showed that the microbiome of obese mice is saturated with enzyme genes that allow for more efficient decomposition of polysaccharides. The intestines of obese mice contained large amounts of fermentation end products – compounds of acetic and butyric acids, which indicates a deeper processing of food components. Calorimetric (from the word "calories"!) analysis of stool samples confirmed this: the stool of ob/ob mice contained fewer calories than in wild-type mice, which did not fully absorb energy from food.
In addition to important information about the "microbial" component of obesity, the authors managed to show the fundamental similarity of the microflora of obese people and mice, which opens up new prospects in the study of the problem of overweight, and possibly solving this problem by "transplanting" healthy microflora or its formation in obese patients.
The fact that the microbiota can control the metabolism of the host is no longer in doubt. Gordon's laboratory's research on the problem of overweight has allowed us to bridge the bridge to the treatment of metabolic diseases, such as general exhaustion affecting children from one to four years old in poor countries with a tropical climate – marasmus (this word has only linguistic relation to marasmus: Greek. marasmos literally means exhaustion, extinction) and kwashiorkor (on the language of one of the tribes of Ghana, kwashiorkor – "red boy"). The occurrence of diseases is associated with a lack of proteins and vitamins during the transition from breastfeeding to adult food. But the disease selectively affects children whose brothers and sisters have not experienced any problems with the transition to a traditional diet for this region. Studies have shown that the intestinal microflora of sick children is strikingly different from the microflora of their parents, as well as from the microflora of healthy brothers and sisters. First of all, there was an almost complete absence of Bacteroidetes in the intestinal population and the dominance of rare species belonging to the types of Proteobacteria and Fusobacteria. After sick children (carefully so as not to overdose!) they were fattened with strenuously protein-rich food, their microbiota became similar to normal, such as in relatives, with a predominance of Bacteroidetes and Firmicutes.
Recent studies have not only radically changed the prevailing ideas about the human intestinal microflora, but also contributed to the emergence of a concept that considers the intestinal microbiota as an additional multicellular "organ" of the human body. An organ consisting of various cell lines capable of communicating both with each other and with the host organism. An organ that redistributes energy flows, performs important physiological reactions, changes under the influence of the environment and self-regenerates with changes caused by external conditions.
The continuation of the study of the "bacterial organ" can and should lead to an understanding of the laws of its functioning, the disclosure of its subtle connections with the host organism and, as a result, to the emergence of new methods of combating human diseases through targeted treatment of dysfunctions of both components of the meta-organism.
Valery Poroiko, Candidate of Biological Sciences
University of Chicago, Department of General Surgery
Portal "Eternal youth" www.vechnayamolodost.ru
08.04.2008
The journal version of the article was published in Popular Mechanics No. 4-2008