1. In this manuscript by Custodio and coworkers, the authors describe for the first time the antibacterial activity of a T6SS in Neisseria spp. - N. cinerea, which is a commensal of the human respiratory tract. It is well known that T6SSs mediated bacterial killing; however, the fact that strains expressing a type 4 pilus (TfP) are more susceptible to T6SS-mediated killing is a new finding. The N. cinerea T6SS structural cluster is encoded in a plasmid together with six effector/immunity pair. The N. cinerea T6SS is constitutively active in the conditions tested. The commensal N. cinerea outcompete pathogenic Neisseria species, such as N. meningitidis and N. gonorrhoeae, in co-cultures. The authors also describe that the presence of a capsule can protect prey cells against T6SS attack, and that the expression of Tfp is a disadvantage to the prey cells. The article is well written and contains all the proper controls. I have some comments. 1- Line 82: microcolonies instead of micocolonies2- Have the authors tried to mobilize the Neisseria plasmid containing the T6SS to other bacteria? If yes, please briefly describe de results.3- Figure 2. Was the TssB-GFP contraction observed when attacking cells were not in contact with prey cells? Please include a graph showing number of TssB contraction events when attacker cells were or were not in contact with prey cells.4- Figure 4. Have the authors searched the genome of N. cinerea for additional toxin/immunity proteins other than the six pairs contained in the plasmid?5- Figure 5A-B. It was not clear to me whether N. meningitidis and N. gonorrhoeae do not encode a T6SS or whether the T6SSs in these species are not active under the conditions tested. Please clarify. 6- Figure 5A,C. The attacker:prey ratio of 100:1 seems too high. What was the result obtained if the ratio was used 10:1 or 2:1? Please comment and describe results in the manuscript. 7- Figure 6. Can the Tfp from a prey cell induce the attack by an T6SS+ attacker cell in a similar mechanism as the one described as tit-for-tat (Basler 2013 Cell)? Please include a graph showing number of TssB contraction events comparing Tfp+ and Tfp- prey cells. If the Tfp is triggering more T6SS firing events than the explanation for the phenotype might change. Read the original source
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