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Type IV pili (TFPs) are required for bacterial twitching motility and for phage infection in the opportunistic human pathogen Pseudomonas aeruginosa. Here we describe a phage-encoded protein, D3112 protein gp05 (hereafter referred to as Tip, representing twitching inhibitory protein), whose expression is necessary and sufficient to mediate the inhibition of twitching motility. Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/ extension ATPase, at an internal 40-aa region unique to PilB. Tip expression results in the loss of surface piliation. Based on these observations and the fact that many P. aeruginosa phages require TFPs for infection, Tip-mediated twitching inhibition may represent a generalized strategy for superinfection exclusion. Moreover, because TFPs are required for full virulence, PilB may be an attractive target for the development of novel antiinfectives.phage-host interaction | protein-protein interaction | lysogenic conversion | antipathogenics T emperate phages generally lysogenize rather than kill host bacteria, leading to alteration of host traits by the expression of phage-encoded genes (1). Superinfection exclusion is one example of a phage-mediated physiological change, in which a successful lysogenized phage blocks potential competition by another phage. Superinfection exclusion of phages of similar lineage generally involves phage repressors (i.e., homoimmunity) (1, 2). Some temperate phages, however, have developed repressor-independent superinfection exclusion/immunity systems that block phage adsorption or DNA uptake, enabling infected hosts to become resistant to superinfection by phages sharing the same uptake mechanism (1, 3). We previously reported that Pseudomonas aeruginosa temperate phage D3112 blocks infection by a second phage, MP22, but not vice versa. Both D3112 and MP22 require type IV pilus (TFP)-mediated twitching motility for infection (4). This finding is most likely accounted for by the twitching-inhibitory activity unique to D3112 (5). We reasoned that the elucidation of the superinfection exclusion system of phage D3112 may provide insights into the mechanisms of TFP assembly and function. Moreover, because twitching motility is one of the group behaviors critical to biofilm formation and pathogenicity of P. aeruginosa, elucidation of D3112-mediated superinfection exclusion may also be important in the development of new antibacterial therapies. In the present study, we have identified a phage protein for twitching inhibition and its bacterial target. ResultsExpression of D3112 gp05 Is Necessary and Sufficient for Twitching Inhibition. Zegans et al. previously reported that P. aeruginosa swarming motility is inhibited by phage DMS3 and that the inhibition involves the bacterial Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas system present in some P. aeruginosa strains such as PA14 (6). To investigate the potential involvement of the CRISPR/Cas system in twitching inhibition by phage D3112, we first determ...

Non-mammalian infection models have been developed over the last two decades, which is a historic milestone to understand the molecular basis of bacterial pathogenesis. They also provide small-scale research platforms for identification of virulence factors, screening for antibacterial hits, and evaluation of antibacterial efficacy. The fruit fly, Drosophila melanogaster is one of the model hosts for a variety of bacterial pathogens, in that the innate immunity pathways and tissue physiology are highly similar to those in mammals. We here present a relatively simple protocol to assess the key aspects of the polymicrobial interaction in vivo between the human opportunistic pathogens, Pseudomonas aeruginosa and Staphylococcus aureus, which is based on the systemic infection by needle pricking at the dorsal thorax of the flies. After infection, fly survival and bacteremia over time for both P. aeruginosa and S. aureus within the infected flies can be monitored as a measure of polymicrobial virulence potential. The infection takes ~24 h including bacterial cultivation. Fly survival and bacteremia are assessed using the infected flies that are monitored up to ~60 h post-infection. These methods can be used to identify presumable as well as unexpected phenotypes during polymicrobial interaction between P. aeruginosa and S. aureus mutants, regarding bacterial pathogenesis and host immunity.

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KatA is the major catalase required for hydrogen peroxide (H2O2) resistance and acute virulence in Pseudomonas aeruginosa PA14, whose transcription is driven from the promoter (katAp1) located at 155 nucleotide (nt) upstream of the start codon. Here, we identified another promoter (katAp2), the +1 of which was mapped at the 51 nt upstream of the start codon, which was responsible for the basal transcription during the planktonic culture and down-regulated upon H2O2 treatment under the control by the master regulator of anaerobiosis, Anr. To dissect the roles of the dual promoters in conditions involving KatA, we created the promoter mutants for each -10 box (p1m, p2m, and p1p2m) and found that katAp1 is required for the function of KatA in the logarithmic growth phase during the planktonic culture as well as in acute virulence, whereas katAp2 is required for the function of KatA in the stationary phase as well as in the prolonged biofilm culture. This dismantling of the dual promoters of katA sheds light on the roles of KatA in stress resistance in both proliferative and growth-restrictive conditions and thus provides an insight into the regulatory impacts of the major catalase on the survival strategies of P. aeruginosa.