{"id":230067,"date":"2017-01-13T15:54:31","date_gmt":"2017-01-13T17:54:31","guid":{"rendered":"http:\/\/revistapesquisa.fapesp.br\/en\/?p=230067"},"modified":"2017-01-13T15:54:31","modified_gmt":"2017-01-13T17:54:31","slug":"lethal-contact-2","status":"publish","type":"post","link":"https:\/\/revistapesquisa.fapesp.br\/en\/lethal-contact-2\/","title":{"rendered":"Lethal Contact"},"content":{"rendered":"
\"A

Eduardo Cesar<\/span><\/a> A not always peaceful coexistence: colonies of X. citri (yellow)<\/em> cultivated with colonies of E. coli (white)<\/em> and C. violaceum (pink)<\/em>Eduardo Cesar<\/span><\/p><\/div>\n

Published in May 2015<\/a><\/em><\/p>\n

The Xanthomonas citri<\/em> bacterium causes citrus canker, a disease that has returned to spread through S\u00e3o Paulo plantations. X. citri<\/em> spends only part of its life inside the leaves and fruits of citrus trees. There, protected by an abundant food source, the bacterium multiplies and stimulates the proliferation of plant cells, generating noticeable dark lesions that break apart to release the bacterium into the air. Most of the time, however, these bacteria face conditions that are less hospitable. In the soil or on the outside of leaves, where they are generally found, they face fierce competition with other microorganisms for space and nutrients. Despite this, X. citri<\/em> generally thrives, as seen in Florida orange trees in the United States, which experienced a 50% decrease in yield in recent years due to the spread of citrus canker and another disease known as greening.<\/p>\n

Thousands of years of evolution have prepared the bacterium to cope with its potential competitors. Its rod-like cells are covered with ultrathin filaments that resemble fine hairs. These structures are part of a defense mechanism that destroys other bacteria. Biochemist Shaker Chuck Farah and his team at the University of S\u00e3o Paulo Chemistry Institute (IQ-USP) have demonstrated that by using a specific type of these filaments, X. citri<\/em> is able to release a veritable cocktail of toxic compounds into its potential competitors.<\/p>\n

The hair-like filaments are actually channels\u2013there are at least six known types\u2013that link the internal medium of the bacteria with the external milieu. One particular type of these channels, known as the type IV secretion system (T4SS), consists of more than 100 proteins and is shaped like a needle. It is well known that many types of bacteria use the T4SS to exchange genetic material with other bacteria of the same or different species through a phenomenon known as conjugation, which allows the horizontal transfer of antibiotic resistance genes. At least one bacterium, Agrobacterium tumefaciens<\/em>, transfers DNA through the T4SS to its host, a plant in which it causes tumors known as galls. It is also through this secretion system that some animal and human pathogenic bacteria inject proteins that help them colonize their host. However, little is known about the function of the T4SS in X. citri<\/em> and in the dozens of species that constitute the family Xanthomonadaceae, which includes the genus Stenotrophomonas<\/em> and the species S. maltophilia<\/em>, an opportunistic human pathogen.<\/p>\n

Previous studies have indicated that the T4SS channels in the Xanthomonadaceae family are different from those found in other groups of bacteria. Farah and his team also determined that in X. citri<\/em>, the T4SS does not play an essential role in plant infection. Recently, USP researchers confirmed that the T4SS in these bacteria is used to inject nearly a dozen different toxic proteins (toxins) into other bacteria.<\/p>\n

These toxins digest sugars, proteins and lipids in the walls of competing bacteria, causing them to expel their contents in such a way that the bacteria appear to explode under the microscope. In Farah\u2019s laboratory, biologists Diorge Souza and Gabriel Oka cultured millions of X. citri<\/em> cells with a similar number of Escherichia coli<\/em>, a bacterium normally found in the intestines of mammals, and filmed the culture over time. Frequently, when X. citri<\/em> came in contact with the surface of an E. coli<\/em>, the latter\u2019s cell wall disintegrated, releasing cellular contents, as seen in a video available on the Internet [https:\/\/youtu.be\/0cSXyd9bd7Q]. \u201cThe bacterium becomes deformed when the integrity of its walls is compromised,\u201d Farah explains. \u201cIt\u2019s like a water balloon that pops,\u201d he says.<\/p>\n

The secretion of toxins is activated by contact; however, it remains unclear how Xanthomonas<\/em> recognizes the other species. The bacterium itself, however, is protected from the compounds it produces. Souza and Oka determined that Xanthomonas<\/em> synthesizes antidotes against its toxins. \u201cThe antitoxins are distributed across the wall of Xanthomonas<\/em>,\u201d Souza explains. \u201cThis is what probably prevents it from suffering any damage.\u201d<\/p>\n

 <\/p>\n

Protein attraction<\/strong>
\nIn this regard, one of these antitoxins offered Souza his first clue years ago about the role of T4SS in Xanthomonas<\/em>. In 2005, chemist Marcos Alegria, Farah\u2019s doctoral student at the time, published a study showing that in X. citri<\/em>, a specific protein of this secretion system, VirD4, attracted other proteins, whose functions were unknown at the time, to the channel. One of these proteins, which was designated Xac2609, interacted with protein Xac2610, whose function was also unknown. Later, after determining the three-dimensional structure of Xac2610, Souza began searching public databases for other proteins with similar structures that may provide insights into the function of the T4SS proteins.<\/p>\n

\"Xanthomonas\"<\/a>The first one he found was a protein that blocks the actions of lysozymes and works like an antitoxin. This finding suggested that the interacting partner of Xac2610, Xac2609, was a lysozyme, a protein capable of digesting the chain of sugars on the bacterial wall. After confirming the actions of these two proteins, Souza identified 13 other potential toxins and seven antitoxins encoded in the genome of X. citri<\/em>, in addition to hundreds of other toxins associated with the T4SS in other species of the Xanthomonadaceae family.<\/p>\n

Tests performed on two different species of bacteria, Micrococcus luteus<\/em> and Bacillus subtilis<\/em>, confirmed that the protein encoded by Xac2609 degrades the bacterial wall and that its effect is nullified by Xac2610, as reported in a paper published in March 2015 in the journal Nature Communications<\/em>. However, it remained to be determined whether Xac2609 and the other toxins were the same as those exported by the T4SS. Souza and Oka then developed genetically modified X. citri<\/em> that did not produce the T4SS and cultured these bacteria with E. coli<\/em>, which multiplies more quickly. E. coli<\/em> duplicates itself every 30 minutes, whereas X. citri<\/em> duplication takes approximately five times longer.<\/p>\n

Without the secretor channel, Xanthomonas<\/em> was at a disadvantage. The experiment began with similar numbers of the two species and ended with E. coli dominating the colony. Although it reproduced more slowly, Xanthomonas<\/em> re-established dominance by eliminating competitors when its ability to produce the T4SS was restored. \u201cThe system gives Xanthomonas<\/em> a competitive edge,\u201d Souza says.<\/p>\n

Although E. coli<\/em> does not compete with Xanthomonas<\/em> in nature, the researchers believe that what they observed in the laboratory may also occur in the field. They repeated the test using four other species of Gram-negative bacteria, which, like E. coli<\/em>, have a cellular envelope comprising of three layers: two membranes and a fortified periplasm, composed of a polymer (peptidoglycan) mixed with sugars and amino acids. \u201cTo date, Xanthomonas<\/em> has killed all of them,\u201d says Farah, who began studying the bacteria 15 years ago when he joined the group that sequenced the Xanthomonas<\/em> genome.<\/p>\n

Hostile environment<\/strong>
\nFarah and his team have proof that X. citri<\/em> is armed with the T4SS when it is found on the outside of a leaf, a potentially more hostile environment. \u201cThis mechanism should help the bacteria become more competitive,\u201d says researcher Marcos Antonio Machado from the Sylvio Moreira Citriculture Center in Cordeir\u00f3polis. \u201cTechnologically, this observation makes it possible to look for compounds capable of inhibiting the functioning of this system,\u201d the researcher says. He is studying ways to increase the susceptibility of X. citri<\/em> to compounds such as copper oxychloride, which is used to fight citrus canker in the S\u00e3o Paulo orange groves.<\/p>\n

Farah believes that a better understanding of the T4SS of Xanthomonas<\/em> is important for understanding how bacteria from different species compete with each other when they find themselves in the same environment and use the same resources. \u201cThis competition may have implications on the evolution of both antagonistic and cooperative behaviors between bacterial species,\u201d he says. These studies may also lead to the identification of new toxins and molecular targets for antibacterial drugs. \u201cWe\u2019re using Xanthomonas<\/em>,\u201d adds Farah, \u201cto understand the universal functions of bacteria.\u201d<\/p>\n

Project<\/strong>
\nCyclic di-GMP signaling and the type IV macromolecule secretion system in Xanthomonas citri<\/em> (n\u00ba 2011\/07777-5<\/a>); Grant mechanism<\/strong> Thematic Project; Principal investigator <\/strong>Shaker Chuck Farah (IQ-USP); Investment<\/strong> R$2,146,849.71 (FAPESP \u2013 for the entire project).<\/p>\n

Scientific articles<\/em>
\nSOUZA, D. P. et al<\/em>. Bacterial killing via a type IV secretion system<\/a>. Nature Communications<\/strong>. March 6, 2015.
\nSOUZA, D. P. et al<\/em>. A component of the Xanthomonadaceae type IV secretion system combines a VirB7 Motif with a no domain found in outer membrane transport proteins<\/a>. PLOS Pathogens<\/strong>. 2011.
\nALEGRIA, M. C. et al<\/em>. Identification of new protein-protein interactions involving the products of the chromosome- and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. citri<\/em><\/a>. Journal of Bacteriology<\/strong>. V. 187, p. 2315-25. 2005.<\/p>\n","protected":false},"excerpt":{"rendered":"Structure allows bacterium to release toxic compounds into rivals","protected":false},"author":16,"featured_media":0,"comment_status":"open","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"_acf_changed":false,"_exactmetrics_skip_tracking":false,"_exactmetrics_sitenote_active":false,"_exactmetrics_sitenote_note":"","_exactmetrics_sitenote_category":0,"footnotes":""},"categories":[159],"tags":[211],"coauthors":[105],"class_list":["post-230067","post","type-post","status-publish","format-standard","hentry","category-science","tag-biochemistry"],"acf":[],"_links":{"self":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/230067","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/users\/16"}],"replies":[{"embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/comments?post=230067"}],"version-history":[{"count":0,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/posts\/230067\/revisions"}],"wp:attachment":[{"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/media?parent=230067"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/categories?post=230067"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/tags?post=230067"},{"taxonomy":"author","embeddable":true,"href":"https:\/\/revistapesquisa.fapesp.br\/en\/wp-json\/wp\/v2\/coauthors?post=230067"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}