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Lethal Contact

A 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 with colonies of E. coli (white) and C. violaceum (pink)

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

Published in May 2015

The Xanthomonas citri bacterium causes citrus canker, a disease that has returned to spread through São Paulo plantations. X. citri 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 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.

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 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 hair-like filaments are actually channels–there are at least six known types–that 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, 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 and in the dozens of species that constitute the family Xanthomonadaceae, which includes the genus Stenotrophomonas and the species S. maltophilia, an opportunistic human pathogen.

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, 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.

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’s laboratory, biologists Diorge Souza and Gabriel Oka cultured millions of X. citri cells with a similar number of Escherichia coli, a bacterium normally found in the intestines of mammals, and filmed the culture over time. Frequently, when X. citri came in contact with the surface of an E. coli, the latter’s cell wall disintegrated, releasing cellular 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 contact; however, it remains unclear how Xanthomonas recognizes the 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 Xanthomonas,” Souza explains. “This is what probably prevents it from suffering any damage.”


Protein attraction
In this regard, one of these antitoxins offered Souza his first clue years ago about the role of T4SS in Xanthomonas. In 2005, chemist Marcos Alegria, Farah’s doctoral student at the time, published a study showing that in X. citri, 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.

XanthomonasThe 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, in addition to hundreds of other toxins associated with the T4SS in other species of the Xanthomonadaceae family.

Tests performed on two different species of bacteria, Micrococcus luteus and Bacillus subtilis, 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. 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 that did not produce the T4SS and cultured these bacteria with E. coli, which multiplies more quickly. E. coli duplicates itself every 30 minutes, whereas X. citri duplication takes approximately five times longer.

Without the secretor channel, 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, Xanthomonas re-established dominance by eliminating competitors when its ability to produce the T4SS was restored. “The system gives Xanthomonas a competitive edge,” Souza says.

Although E. coli does not compete with Xanthomonas 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, 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. “To date, Xanthomonas has killed all of them,” says Farah, who began studying the bacteria 15 years ago when he joined the group that sequenced the Xanthomonas genome.

Hostile environment
Farah and his team have proof that X. citri is armed with the 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. “Technologically, this observation makes it possible to look for compounds capable of inhibiting the functioning of this system,” the researcher says. He is studying ways to increase the susceptibility of X. citri to compounds such as copper oxychloride, which is used to fight citrus canker in the São Paulo orange groves.

Farah believes that a better understanding of the T4SS of Xanthomonas 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. “This competition may have implications on the evolution of both antagonistic and cooperative behaviors between bacterial species,” he says. These studies may also lead to the identification of new toxins and molecular targets for antibacterial drugs. “We’re using Xanthomonas,” adds Farah, “to understand the 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.