Changing the Paradigm for Management of Pediatric Primary Spontaneous Pneumothorax: A Simple Aspiration Test Predicts Need for Operation

Purpose Chest tube (CT) management for pediatric primary spontaneous pneumothorax (PSP) is associated with long hospital stays and high recurrence rates. To streamline management, we explored simple aspiration as a test to predict need for surgery. Methods A multi-institution, prospective pilot study of patients with first presentation for PSP at 9 children’s hospitals was performed. Aspiration was performed through a pigtail catheter, followed by 6 h observation with CT clamped. If pneumothorax recurred during observation, the aspiration test failed and subsequent management was per surgeon discretion. Results Thirty-three patients were managed with simple aspiration. Aspiration was successful in 16 of 33 (48%), while 17 (52%) failed the aspiration test and required hospitalization. Twelve who failed aspiration underwent CT management, of which 10 (83%) failed CT management owing to either persistent air leak requiring VATS or subsequent PSP recurrence. Recurrence rate was significantly greater in the group that failed aspiration compared to the group that passed aspiration [10/12 (83%) vs 7/16 (44%), respectively, P = 0.028]. Conclusion Simple aspiration test upon presentation with PSP predicts chest tube failure with 83% positive predictive value. We recommend changing the PSP management algorithm to include an initial simple aspiration test, and if that fails, proceed directly to VATS

Immunity function and Dienes type for site-directed PefE mutants.

<p>Immunity function and Dienes type for site-directed PefE mutants.</p

Subtle variation within conserved effector operon gene products contributes to T6SS-mediated killing and immunity

<div><p>Type VI secretion systems (T6SS) function to deliver lethal payloads into target cells. Many studies have shown that protection against a single, lethal T6SS effector protein requires a cognate antidote immunity protein, both of which are often encoded together in a two-gene operon. The T6SS and an effector-immunity pair is sufficient for both killing and immunity. HereIn this paper we describe a T6SS effector operon that differs from conventional effector-immunity pairs in that eight genes are necessary for lethal effector function, yet can be countered by a single immunity protein. In this study, we investigated the role that the PefE T6SS immunity protein plays in recognition between two strains harboring nearly identical effector operons. Interestingly, despite containing seven of eight identical effector proteins, the less conserved immunity proteins only provided protection against their native effectors, suggesting that specificity and recognition could be dependent on variation within an immunity protein and one effector gene product. The variable effector gene product, PefD, is encoded upstream from <i>pefE</i>, and displays toxic activity that can be countered by PefE independent of T6SS-activity. Interestingly, while the entire <i>pef</i> operon was necessary to exert toxic activity via the T6SS in <i>P</i>. <i>mirabilis</i>, production of PefD and PefE alone was unable to exert this effector activity. Chimeric PefE proteins constructed from two <i>P</i>. <i>mirabilis</i> strains were used to localize immunity function to three amino acids. A promiscuous immunity protein was created using site-directed mutagenesis to change these residues from one variant to another. These findings support the notion that subtle differences between conserved effectors are sufficient for T6SS-mediated kin discrimination and that PefD requires additional factors to function as a T6SS-dependent effector.</p></div

Strains and plasmids.

<p>Strains and plasmids.</p

Immunity function and Dienes type for chimeric PefE:PefE2 proteins.

<p>Immunity function and Dienes type for chimeric PefE:PefE2 proteins.</p

Chimeric immunity proteins constructed from PefE and PefE2 reveal amino acid residues responsible for specificity.

<p><b>(A)</b><i>P</i>. <i>mirabilis</i> HI4320 <i>pefE</i> (black) and <i>pefE2</i> (grey) were PCR-amplified to construct chimeric immunity proteins to determine regions within PefE and PefE2 responsible for immunity function. <b>(B)</b> Nine chimeras (C1-C9) composed of partial gene fragments from <i>pefE</i> and <i>pefE2</i> were created using conserved restriction sites (XbaI, BamHI, NspI, EcoRI, and EcoRV) located within the genes and ligated into pBAD-MycHisA. The amino acid sequence number of the 3’ end <i>pefE</i> or <i>pefE2</i> gene fragment is indicated below each restriction enzyme site and also depicts the point of fragment ligation. <b>(C)</b> Following induction by 10 mM L-arabinose, 9C1 expressing <i>pefE</i> restored immunity function against parent strain HI4320 (no Dienes line) in contrast to 9C1 containing pBAD empty vector or 9C1 expressing <i>pefE2</i>, which were unable to complement the immunity defect and thus result in formation of a Dienes line. Chimeric immunity proteins C2, C3, C4, C7, and C9 restored immunity against HI4320 (no Dienes line; white arrows). <b>(D)</b> Amino acid sequences of residues 130–150 from PefE and PefE2 are highly conserved except for residues 137, 138, and 140 suggesting that these residues are responsible for specificity of immunity.</p

PefD and PefE can function as a toxin/anti-toxin independent of T6SS activity.

<p><b>(A)</b> Introduction of the entire <i>pef</i> operon from strain HI4320 into <i>P</i>. <i>mirabilis</i> BB2000 is sufficient to form a Dienes line with wild-type parental strain BB2000 (black arrow), while PefD and PefE alone are not sufficient for T6SS-dependent effector activity in <i>P</i>. <i>mirabilis</i> BB2000 and produce a result identical to the lack of a boundary between isogenic swarms of wild-type BB2000 (white arrows). <b>(B)</b> Serial dilutions of <i>E</i>. <i>coli</i> containing PefD and PefE, or the predicted nuclease domain of PefD (PefD-Tox) and PefE, on LB agar (uninduced), agar containing IPTG to induce PefD or PefD-Tox, and agar containing IPTG and L-arabinose to additionally induce PefE. Toxicity is observed when PefD or PefD-Tox are induced in the absence of PefE induction.</p

Comparison of protein sequences of PefE2 of <i>P</i>. <i>mirabilis</i> HI4320 and PefE of BA6163 shows high level of conservation.

<p><b>(A)</b> Amino acid sequence alignments indicate that PefE of BA6163 is more similar to PefE2 than PefE of HI4320 with 315/320 shared amino acids (98% identity). Amino acids shaded in red indicate residue similarities between BA6163 PefE and HI4320 PefE2 that differ from PefE of HI4320. <b>(B)</b> Arrows represent the gene products of the primary effector operon in HI4320 and BA6163. Percent identity of the amino acid sequences comparison and number of conserved amino acids are listed for each protein. PefE (shaded black) has the lowest percent identity, 89.6%, of the two strains. <b>(C)</b> Following induction on 10 mM L-arabinose, 9C1 containing the BA6163 <i>pef</i> operon (Pef_BA) was unable to complement 9C1 immunity resulting in a visible Dienes line (black arrow) with parent strain, HI4320, while 9C1 containing the HI4320 <i>pef</i> operon (Pef_HI) complements immunity against HI4320 (white arrow). The boxed area in (C) indicates that 9C1 containing Pef_HI and Pef_BA are restored for killing and are not immune to one another.</p

<i>P</i>. <i>mirabilis</i> HI4320 encodes two functionally distinct PefE proteins.

<p><b>(A)</b> The primary effector operon; <i>hcp</i>, <i>vgrG</i>, <i>pefABCDEFG</i>, in HI4320 (grey arrows). Downstream genes from the <i>pef</i> operon include a second copy of <i>pefE (pefE2</i>, black arrow) located next to two transposase pseudogenes (PMI0759 and PMI0760) and a recombination hot-spot (<i>rhs</i>) gene fragment (PMI0761) depicted as blue arrows. <b>(B)</b> Alignment of HI4320 PefE and PefE2 predicted amino acid sequences. Amino acids shaded in red indicate residue similarities (284/320 amino acids, 89% identity) between HI4320 PefE and PefE2. <b>(C)</b> Dienes line formation is observed under uninduced conditions (-) with 9C1 containing empty vector, 9C1 containing <i>pefE</i>, and 9C1 containing <i>pefE2</i> against parent strain, HI4320. Following induction on 10 mM L-arabinose (+), expression of <i>pefE2</i> in 9C1 does not complement the immunity defect caused by disruption of <i>pefE</i> (black arrow) against parent strain HI4320. Only <i>pefE</i> expressed in 9C1 restores immunity (white arrow) against HI4320. <b>(D)</b> Western blot of 6xHIS tagged PefE and 6xHIS tagged PefE2 expressed during growth in LB broth with (+) and without (-) 10 mM L-arabinose.</p