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Lethal Contact

Needle-shaped structure allows the Xanthomonas citri bacterium to release toxic compounds into rival microorganisms

A not always peaceful coexistence: colonies of X. citri (yellow) cultivated in colonies of E. coli (white) and C. violaceum (pink)

Eduardo CesarA not always peaceful coexistence: colonies of X. citri (yellow) cultivated in colonies of E. coli (white) and C. violaceum (pink)Eduardo Cesar

The Xanthomonas citri bacterium, which causes citrus canker, a disease that has returned to spread through São Paulo plantations, spends only part of its life inside the leaves and fruit of citrus trees. There, protected by an abundant food source, it multiplies and stimulates the proliferation of plant cells, generating noticeable dark lesions that, when breaking apart, dissipate into the air. Most of the time, however, the conditions these bacteria face are much less hospitable. In soil or on the outside of the leaves, where they are generally found, they face fierce competition with other microorganisms for space and nutrients. Despite this, Xanthomonas citri generally do well, as we have seen with Florida oranges 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.

Thousands of years of evolution have prepared the bacterium to deal with its potential competitors. Its rod-like cells are covered with ultra-thin 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 São Paulo Chemistry Institute (IQ-USP) have demonstrated that by using a specific type of these filaments, X. citri is able to release a veritable cocktail of toxic compounds into its potential competitors.

The filaments that resemble hairs are actually channels – there are at least six known types – that link the internal medium of the bacteria to the outside. One particular variety 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 already known that many types of bacteria use it to exchange genetic material with other bacteria of the same or other species through a phenomenon known as conjugation, which allows the horizontal transfer of genes associated with the development of antibiotic resistance. At least one bacterium, Agrobacterium tumefaciens, transfers DNA through the T4SS to its host, a plant, in which it causes tumors known as branches. It is also through this secretion system that some species associated with diseases in animals and humans inject proteins that help them colonize the host. But we did not know the function of the T4SS in the Xanthomonas citri and in the dozens of species that make up the family Xanthomonadaceae, which include bacteria of the genus Stenotrophomonas – among them, the species S. maltophilia, an opportunistic pathogen in humans.

Previous studies have indicated that in the Xanthomonadaceae family, the T4SS channels were different from those found in other groups of bacteria. Farah and his team had also determined that in the case of Xanthomonas citri, that structure did not play an essential role in plant infection. Now the USP researchers have verified that the T4SS in these bacteria is used to inject nearly a dozen different toxic proteins (toxins) into other bacteria.

With an intact toxin secretion system, Xanthomonas citri bacteria (blue) dominate the colony; without it, X. citri is at a disadvantage and loses ground to Escherichia coli (yellow)

Diorge Douza / USPWith an intact toxin secretion system, Xanthomonas citri bacteria (blue) dominate the colonyDiorge Douza / USP

These toxins digest sugars, proteins and lipids from the walls of competing bacteria, causing them to expel their contents in such a way that sometimes appears explosive under the microscope. In Farah’s laboratory, biologists Diorge Souza and Gabriel Oka placed millions of X. citri cells to live with a similar number of Escherichia coli, the bacteria normally found in the intestines of mammals, and filmed what happened. Many of the times in which the X. citri touched the surface of an E. coli, its wall broke apart, pouring out its contents as seen in a video available on the Internet []. “The bacterium becomes deformed when the integrity of its walls is compromised,” Farah explains. “It’s like a water balloon that pops,” he says.

The secretion of toxins is activated by the contact, although it is still not known exactly how the Xanthomonas recognizes the bacteria of other species. The bacterium itself, however, is protected from the compounds it produces. Souza and Oka determined that Xanthomonas synthesizes antidotes against its toxins. “The antitoxins are distributed across the wall of the Xanthomonas,” Souza explains. “This is what probably prevents it from suffering any damage.”

Protein attraction
In this regard, it was one of these antitoxins that offered Souza his first clue years ago about the role the Type IV secretion system plays in Xanthomonas. In 2005, Farah’s doctoral student at the time, chemist Marcos Alegria, had published a study showing that in X. citri a specific protein – VirD4 – of this secretion system attracted other proteins, all with a function unknown at the time, to the channel. One of these proteins, which was given the abbreviation Xac2609, interacted with protein Xac2610, whose function was also unknown. Some time later, after determining the three-dimensional structure of the Xac2610, Souza began searching the public databases for other proteins with similar structures that might be able to indicate their function.

The first one he found was a protein that blocks the action of lysozyme and works like an antitoxin. This finding suggested that the interacting partner of the Xac2610, the Xac2609, could be a lysozyme, a protein capable of digesting the chain of sugars on the bacterial wall. After confirming the action of these two proteins, Souza identified other potential toxins and antitoxins – in all, 13 of the former and seven of the latter – encoded in the genome of Xanthomonas citri, in addition to hundreds of other toxins associated with the Type IV secretion system of other species of the Xanthomonadaceae family.

Tests run on two different species of bacteria, Micrococcus luteus and Bacillus subtilis, confirmed that the protein encoded by the Xac2609 degrades the bacterial wall and that its effect is nullified by the Xac2610, according to a paper published in March 2015 in the journal Nature Communications. But it still needed to be determined whether this and other toxins were the same as those exported by the T4SS. Souza and Oka then developed genetically modified X. citri that did not produce the T4SS and placed them to grow together with E. coli bacteria, which multiply more quickly – E. coli duplicates itself every 30 minutes while it takes Xanthomonas citri up to five times more time to do so.

without it, X. citri is at a disadvantage and loses ground to Escherichia coli (yellow)

Diorge Douza / USPWithout it, X. citri is at a disadvantage and loses ground to Escherichia coli (yellow)Diorge Douza / USP

Without the secretor channel, the Xanthomonas 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, the Xanthomonas went back to dominating, eliminating the competitors when the researcher gave it back its ability to produce the T4SS. “The system gives the Xanthomonas a competitive edge,” Souza says.

Although E. coli does not compete with Xanthomonas in nature, the researchers believe that what they saw in the laboratory could also be the case in the field. They repeated the test using four other species of bacteria classified as gram-negative, which, like E. coli, have a cellular envelope made up of three layers – two membranes and a fortified periplasm, composed of a polymer (peptideoglycan) mixed with sugars and amino acids. “Up to now, the Xanthomonas has killed all of them,” says Farah, who began studying the bacteria 15 years ago when he joined the group that sequenced the genome of the Xanthomonas.

Hostile environment
Farah and his team have proof that the X. citri is armed especially with T4SS when it is found on the outside of a leaf, a potentially more hostile environment. “This mechanism should help the bacteria become more competitive,” says researcher Marcos Antonio Machado, from the Sylvio Moreira Citriculture Center in Cordeirópolis. “In technological terms, this determination opens the door to the possibility that we can look for compounds that are capable of inhibiting the functioning of this system,” the researcher says. He is studying ways to increase the susceptibility of the X. citri to compounds such as copper oxychloride, used to fight citrus canker in the São Paulo orange groves.

Farah believes that a better understanding of the IV secretion system of the Xanthomonas is important for learning how bacteria from different species compete with each other when they find themselves in the same environment and are using the same resources. “This competition may have implications on the evolution of behaviors that are both antagonistic and cooperative between bacterial species,” he says. These studies could also lead to the identification of new toxins and molecular targets for drugs with antibacterial action. “We’re using the Xanthomonas,” adds Farah, “to understand the more universal functions of bacteria.”

Cyclic di-GMP signaling and the type IV macromolecule secretion system in Xanthomonas citri (No. 2011/07777-5); Grant mechanism Thematic Project; Principal investigator Shaker Chuck Farah (IQ-USP); Investment R$2,146,849.71 (FAPESP – for the entire project).

Scientific articles
SOUZA, D. P. et al. Bacterial killing via a type IV secretion system. Nature Communications. March 6, 2015.
SOUZA, D. P. et al. A component of the Xanthomonadaceae type IV secretion system combines a VirB7 Motif with a no domain found in outer membrane transport proteins. PLOS Pathogens. 2011.
ALEGRIA, M. C. et al. 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. Journal of Bacteriology. V. 187, p. 2315-25. 2005.