Cytohesin-1 and -3 are not required for LCV biogenesis

In section 3.2.1 the requirement of cytohesin GEF activity in the efficient invasion and propagation of L. pneumophila in BMDM in vitro was examined. Experiments in this section aimed to elucidate whether cytohesin-1 or cytohesin-3 are im-portant host factors for LCV biogenesis in phagocytes in vivo (287). The method utilizes the effector protein, RalF, that is translationally fused to a -lactamase reporter (BlaM). The form of BlaM utilised lacks a signal peptide for secretion and hence is only secreted into the host cell cytosol by L. pneumophila if fused to an effector protein. Cytosolic BlaM can be detected with the substrate, CCF2. In the presence of BlaM, CCF2 is cleaved into two products which leads to a shift in the fluorescence emission of CCF2 from green to blue (see section 2.6.1.8).

Mice were infected with different L. pneumophila strains including L. pneumophila

flaA transformed with pXDC61 expressing RalF-BlaM, or the control strains L.

pneumophila flaA, L. pneumophila flaA transformed with empty pXDC61 vec-tor and a L. pneumophila flaAdotA double mutant transformed with pXDC61 expressing RalF-BlaM. L. pneumophila flaAdotA expresses RalF-BlaM but lacks a functional Dot/Icm secretion system, and so cannot translocate effectors into phagocytes. One day after infection, cells were collected from the BAL and stained with CCF2 to analyse the translocation of bacterial effectors into individ-ual phagocytes identified using antibodies to cell specific markers.

Figure 3.3 shows the translocation of RalF-BlaM in AM of Cyth1^-/- and Cyth3^-/- as well as WT mice. Mice that were infected with L. pneumophila flaA with pXDC61 RalF-BlaM displayed a conversion of CCF2 from green to blue in AM (Figure 3.3A), whereas AM from mice that have received the dotA mutant presented no conversion of CCF2 to blue, emphasizing that translocation of L. pneumophila effector proteins depends on a functional Dot/Icm type IV secretion system (Figure 3.3B). Also, no significant conversion was found in other controls for cells

infected with L. pneumophila flaA or L. pneumophila flaA transformed with empty pXDC61 vector (not shown).

In AM from WT mice the percentage of CCF2 blue-converted cells accounted for 8.1% in average which was in the range that has been published previously (288).

In Cyth1^-/- and Cyth3^-/- no significant difference was found in the proportions of CCF2 blue-converted AM compared to WT AM. (Figure 3.3C).

Consequently, Cyth1 and Cyth3 had no influence on the translocation of RalF into AM in vivo.

Translocation of al laM in AM

CC blue in AM

A

C B

T h h

Figure 3.3: Translocation of L. pneumophila effectors in cytohesin-1 or -3 deficient alveolar macrophages in vivo. Mice were infected with L. pneumoph-ila expressing the translocated effector RalF fused to the reporter protein BlaM (pXDC61 RalF-BlaM) or respective control strains for 1 day. Cells were collected by bronchoalveolar lavage and stained with CCF2-AM dye, which is cleaved by translocated RalF-BlaM resulting in a spectral shift from green to blue. A. Gating strategy to identify alveolar macrophages with cleaved CCF2 (blue). Number rep-resents percentage of AM with translocated RalF as measured by CCF2-blue fluorescence. B. Cells from mice infected with control L. pneumophila flaAdotA strain containing pxDC61 plasmid expressing RalF-BlaM shows no translocation.

C. Percentage of AM with translocated RalF-BlaM measured by CCF2-blue fluo-rescence from C57BL/6 (WT), cytohesin-1 (Cyth1^-/-) and cytohesin-3 (Cyth3^-/-) mice. Graphs present the mean with SEM. n=3 from one experiment. No signifi-cant difference was found between wildtype and knockout mice (threshold p<0.05; unpaired two-tailed student t-test).

Although AM are considered to be the key phagocyte that supports the intracel-lular replication of L. pneumophila, the pathogen is also able to translocate effec-tor proteins into neutrophils, although they do not appear to support intracellular replication (288). Similar to AM, translocation of L. pneumophila effectors into neutrophils depended on the formation of a functional Dot/Icm type IV secretion system representing 2% of CCF2 Blue-converted neutrophils infected with L. pneumophila flaA carrying pXDC61 encoding RalF-BlaM (Figure 3.4A) and 0% of CCF2 conversion in neutrophils infected with L. pneumophila flaAdotA expressing RalF-BlaM (Figure 3.4B).

The percentage of neutrophils that converted to CCF2 blue did not statistically differ among Cyth1^-/-, Cyth3^-/- and WT neutrophils (Figure 3.4C) indicating that Cyth1 and Cyth3 were not required for the translocation of RalF by L. pneumoph-ila in neutrophils.

Translocation of al laM in neutrophils

CC blue in neutrophils

A

C B

T h h

Figure 3.4: Translocation of L. pneumophila effectors in cytohesin-1 or -3 deficient neutrophils in vivo. Mice were infected with L. pneumophila flaA ex-pressing the translocated effector RalF fused to the reporter protein BlaM (pXDC61 RalF-BlaM) or respective control strains for 1 day. Cells were collected by bronchoalveolar lavage and stained with CCF2-AM dye, which is cleaved by translocated RalF-BlaM resulting in a spectral shift from green to blue. A. Gating strategy to identify neutrophils with cleaved CCF2 (blue). Number represents per-centage of neutrophils with translocated RalF as measured by CCF2-blue fluo-rescence. B. Cells from mice infected with control L. pneumophila flaAdotA strain containing pxDC61 plasmid expressing RalF-BlaM shows no translocation.

C. Percentage of neutrophils with translocated RalF-BlaM measured by CCF2-blue fluorescence from C57BL/6 (WT), cytohesin-1 (Cyth1^-/-) and cytohesin-3 (Cyth3^-/-) mice. Graphs present the mean with SEM. n=3 from one experiment.

No significant difference was found between wildtype and knockout mice (thresh-old p<0.05; unpaired two-tailed student t-test).

The blue conversion of CCF2 by translocated RalF- BlaM in MCs was also inves-tigated. One day after infection, few MCs had infiltrated the bronchoalveolar space. However, mice that were infected with L. pneumophila flaA expressing RalF-BlaM showed similar translocation levels in MCs as neutrophils (Figure 3.5A). MCs from mice infected with L. pneumophila flaAdotA mutant did not show any shift in CCF2 green to blue emission (Figure 3.5B).

As in previous analyses, no statistically significant difference was observed in the translocation of RalF-BlaM into MCs in Cyth1 or Cyth3 deficient mice (Figure 3.5C). Therefore, Cyth1 and Cyth3 deficiency did not alter the translocation of RalF-BlaM in MCs.

T h h

Translocation of al laM in MCs

CC blue in MCs

A

C

B

Figure 3.5:Translocation of L. pneumophila effectors in cytohesin-1 or -3 deficient monocyte-derived cells in vivo. Mice were infected with L. pneu-mophila flaA expressing the translocated effector RalF fused to the reporter pro-tein BlaM (pXDC61 RalF-BlaM) or respective control strains for 1 day. Cells were collected by bronchoalveolar lavage and stained with CCF2-AM dye, which is cleaved by translocated RalF-BlaM resulting in a spectral shift from green to blue.

A. Gating strategy to identify monocyte-derived cells with cleaved CCF2 (blue).

Number represents percentage of MCs with translocated RalF as measured by CCF2-blue fluorescence. B. Cells from mice infected with control L. pneumophila

flaAdotA strain containing pxDC61 plasmid expressing RalF-BlaM shows no translocation. C. Percentage of MCs with translocated RalF-BlaM measured by CCF2-blue fluorescence from C57BL/6 (WT), cytohesin-1 (Cyth1^-/-) and cytohe-sin-3 (Cyth3^-/-) mice. Graphs present the mean with SEM. n=3 from one experi-ment. No significant difference was found between wildtype and knockout mice (threshold p<0.05; unpaired two-tailed student t-test).

In summary, the efficiency of L. pneumophila effector translocation was meas-ured in the main pulmonary phagocytes known to internalise L. pneumophila dur-ing infection in vivo. Neither cytohesin-1 nor cytohesin-3 altered the efficiency of effector translocation and were therefore unlikely to influence LCV biogenesis.

3.2.3 Cytohes3 contributes to weight recovery after L. pneumophila